Generating vascular smooth muscle cells in vitro from ES cells

ABSTRACT

A simplified and inexpensive method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells is provided. The method and the progenitor cells provided herein can be used for in-vitro vascular engineering, treatment of congenital and acquired vascular and hematological abnormalities, for evaluation and development of drugs affecting vasculo- and angiogenic processes, and for further investigation into tissue differentiation and development.

RELATED APPLICATIONS

This Application a divisional of U.S. patent application Ser. No.10/963,834 filed on Oct. 14, 2004, which is a continuation-in-part ofPCT Patent Application No. PCT/IL03/00320 filed on Apr. 15, 2003, whichis a continuation of U.S. patent application Ser. No. 10/211,522 filedon Aug. 5, 2002, now U.S. Pat. No. 7,247,477 issued on Jul. 24, 2007,which claims the benefit of U.S. Provisional Patent Application No.60/372,429 filed on Apr. 16, 2002. The contents of the above-mentionedapplications are hereby incorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel methods for the isolation andculture of vasculogenic progenitor cells from stem cells and, moreparticularly, to methods for use of vasculogenic progenitor cells intissue engineering, research and diagnostics.

Recently, techniques have been developed which allow human embryonicstem cells to proliferate indefinitely in culture, enablingexperimentation with induction of differentiation in a directed,tissue-specific manner (Itskovitz-Eldor, J et al Mol Med 2000; 6:88-95,Reubinoff B E at al Nat Biotech 2000; 18:399-404, Schuldiner M et alPNAS USA 2000; 97:11307-12). Human embryonic stem cell growth anddevelopment is being carefully studied, and the rapidly accumulatingknowledge is being employed in a variety of innovative therapeuticapplications including in-vitro tissue engineering, transplantationmedicine, generation of transgenic embryos and treatment of degenerativedisease. Most significantly, the President of the U.S. has recognizedthe overwhelming importance of embryonic stem cells to medicine andresearch, and has recently sanctioned projects using existing humanembryonic stem cell lines (White House Fact Sheet: Embryonic Stem CellResearch, Aug. 9, 2001). However, in-vitro manipulation of the complexsteps of development, to reliably produce substantial amounts of desiredcell lineages and specific phenotypes remains a crucially importantgoal.

Blood Vessel Formation in Embryonic Development and Adult Life

In the early stages of embryonic development, vessel formation occurs bya process referred to as vasculogenesis, in which mesodermally-derivedendothelial cell progenitors undergo de-novo differentiation, expand andcoalescence to form a network of primitive tubules (Yancopoulos G D etal Nature 2000; 407:242). These blood vessels are generally composed oftwo cell lineages, each serving a different function: internalendothelial cells that form the channels for blood conduction, but alonecannot complete vasculogenesis; and periendothelial smooth muscle cellsthat protect and stabilize the fragile channels from rupture and providehaemostatic control (Carmeliet P Nature Med 2000; 6:389). A third celllineage, the hematopoietic cells, share a common progenitor with thevascular cells, and differentiate into the blood cells. In thevertebrate embryo vasculogenesis occurs in the paraxial and lateralmesoderm, giving rise to the primordia of the heart, the dorsal aorta,and large vessels of the head, lung and gastrointestinal system.Angiogenesis involves the maturation and remodeling of the primitivevascular plexus into a complex network of large and small vessels.Angiogenesis also leads to vascularization of initially avascular organssuch as kidney, brain and limb buds.

Angiogenesis is also required postnatally for normal tissue growth, andcontinues throughout adult life, for example during neo-vascularizationof the endometrium during normal female estrus, during pregnancy in theplacenta, and during wound healing (Risau, et al Nature 1997;386:671-674).

In addition, a number of diseases and disorders have been associatedwith abnormal endothelial growth: endothelial hyperproliferation inatherosclerosis, neovascularization in tumor growth and metastasis, andderegulated angiogenesis in rheumatoid arthritis, retinopathies,hemangiomas and psoriasis (Folkman et al Nature Med. 1995; 1: 27-31;Hanahan and Folkman, Cell 1996; 86:353-64).

Embryonic Endothelial Cells In-Vitro

Research into the functions, origin and nature of embryonic endothelialcells (EEC) has revealed that EECs can promote liver organogenesis(Matsumoto K et al Science 2001; 294:559), induce pancreasdifferentiation (Lammert E et al 2001; 294:564) and trans-differentiateinto cardiac muscle cells under specific conditions (Condorelli G et al2001; 98:10733). While the nature of differentiation and development ofendothelial precursors is not yet fully understood, it is becoming clearthat hematopoietic development and the generation of vascular smoothmuscle cells (v-SMC) are tightly linked with vascular development.

Embryonic stem cells are difficult to maintain in culture, tending tospontaneously differentiate. For ongoing cultures, cells from the innermass of blastocysts are typically grown on a layer of mouse embryonicfibroblast “feeder” cells to preserve their undifferentiated phenotypeand proliferabilty (Keller, G M Curr Opin Cell Biol 1995; 7:862-69). Inmice, early differentiation into embryonically distinct cell types canbe induced by coculture with stromal cell lines (Palacios R, et al PNASUSA 1995; 92:7530-34), culture on substrates such as fibronectin,laminin, collagen, etc. (Ogawa M et alu Blood 1999; 93: 1168-77) or invitro aggregation of embryoid stem (ES) cells into “embryoid bodies”(EB), demonstrating regional differentiation into three germ layers(Keller, G M Curr Opin Cell Biol 1995; 7:862-69).

Murine Embryonic Stem Cells

Study of vasculogenic events in murine ES cells has been instructive.Both hematopoietic and endothelial cells have been observed in blastcell colonies generated from mouse ES cell-derived embryoid bodies (ChoiK, et al Development 1998; 125:725). Also working with murine ES cells,Nishikawa and colleagues demonstrated that 3-D embryoid body formationwas not required for differentiation of lateral mesoderm cells. Whencultured non-aggregated mouse embryonic cells were grown on a collagensubstrate, cells expressing vascular endothelial Cadherin (VE-cad+) werefound to give rise to hematopoietic cells (Nishikawa S I, et alDevelopment 1998; 125:1747, Nishikawa S I et al Immunity 1998; 8:761,and Fujimoto T, et al Genes Cells 2001; 6:1113). Where markers of smoothmuscle cell (SMC) phenotype (e.g. surface markers and morphology) areobserved, early periendothelial SMCs associated with embryonicendothelial tubes can be shown to trans-differentiate from theendothelium (Gittenberger de-Groot, A C et al, Atheroscler Thromb VascBiol 1999; 19:1589), and differentiation of embryonic common vascularprogenitors (Flk1+) into endothelial and smooth muscle cells can beenobserved (Yamashita J et al Nature 2000; 408:92). However, attempts todirectly extrapolate from mouse to human EC systems have met withdisappointing results, indicating that many developmental processes andrequirements are species specific (see, for example, Reubinoff B E etal, Nat. Biotechnolog. 2000; 18:399-404). Specifically, in contrast toit's expression in mouse embryonic stem (mES) cells, the vascularspecific growth factor receptor VEGFR 2 (Flk-1/KDR) is expressed inundifferentiated human embryonic stem cells (hES) (Kaufman, D S et alPNAS USA 2001; 98:10716-21) and does not increase during the first weekof differentiation (Levenberg, S et al PNAS USA 2002; 99:4391-96),indicating that the timing of VEGFR 2 expression may vary amongvertebrate species (also reviewed by Nishikawa; Nishikawa S I et al CurrOpin Cell Biol 2001; 13:862-69). Levenberg et al (Levenberg, S et alPNAS USA 2002; 99:4391-96) further reported that other endothelialmarkers, namely vascular endothelial cadherin (VE-cad) andplatelet-endothelial cell adhesion molecule-1 (PECAM1/CD31), increasedduring the first week of hES differentiation. Clearly, coordination ofexpression of specific endothelial-specific factors, in the appropriatecombinations, are crucial to human vasculogenesis.

Human Embryonic Stem Cells

Human embryonic stem (hES) cell lines were first derived in 1998(Thomson, J A et al Science 1998; 282:1145; U.S. Pat. No. 6,200,806 toThomson et al; U.S. Pat. No. 6,331,406 to Gearhart J D and Shamblott MJ), and have recently been induced to differentiate in vitro in a celllineage-specific manner (Schuldiner M et al PNAS 2000; 97:11307-312,International Patent Application WO0210347 A2 to Benvenisty, N). SincehES cells maintain the embryonic stem cell phenotype throughout hundredsof doubling times, and differentiate to all embryonic cell lineages,they provide a potentially unlimited source of cells for study andclinical application. Both hematopoietic and endothelial celldifferentiation have been observed in human ES cells. To date,hematopoietic differentiation of the hES cells has required coculturingwith either the S17 (murine bone marrow) or C166 (yolk sac endothelial)stromal cell lines, inducing the appearance of primary humanhematopoietic tissue characteristics such as cell surface antigen CD34and hematopoietic colony formation (Kaufman, D S et al PNAS USA 2001;98:10716-21). In another recent study, endothelial cells were selectedby cell sorting (FACS) from human embryoid bodies (EB) using monoclonalantibodies raised against the endothelial-specific marker PECAM-1(Levenberg, S et al PNAS USA 2002; 99:4391-96). The selected, PECAM-1+embryoid body-derived (EBD) cells exhibited endothelial-specificcharacteristics such as von Willebrand factor, VEGFR-2 and VE-cadsurface markers and primitive, vessel-like cord formation when culturedon a soft substrate (Matrigel). PECAM-1+ EBD cells were further observedforming vascular structures in-vivo following seeding on biodegradablepolymer matrix sponges and implantation into SCID mice. However, all ofthe abovementioned methods for differentiation of human ES requireeither coculturing with non-human cells or embryoid body formation priorto appearance of endothelial phenotypes, and immunofluorescent cellsorting for selection according to endothelial cell markers, renderingthem both costly and unsuitable for many clinical applications. Thus, itwould be advantageous to provide a simplified, less expensive method ofculturing, selecting and directing differentiation of human embryonicstem cells, without the limitations of aggregation into embryoid bodiesor immunofluorescent selection.

Prior art discloses a number of techniques and methods for preparationand use of embryonic stem cells for differentiation. Early techniquesrequired inner-cell mass cells from blastocyst-stage embryos (fresh orcryopreserved) as a source of stem cells (see, for example,International Patent Application No. WO 0129206 A1 to Cibelli et al;U.S. Pat. Application Publication Nos. 20020045259 A1 to Lim et al,20020004240 A1 to Wang). Many others rely upon aggregation of the stemcells into embryoid bodies for initiation of differentiation (see, forexample, International Patent Application No. WO 0070021 A3 toItskovitz-Eldor J and Benvenisty N).

Various methods for differentiation of stem cells in culture have alsobeen disclosed. International Patent Application No. WO 0134776 A1, U.S.Pat. Application Publication No. 20020015694 A1, and U.S. Pat. No.6,280,718, all to Kaufman, D et al, disclose methods of differentiatinghuman embryonic stem cells into hematopoietic cells by coculture withmammalian stromal cells. U.S. Patent Application Publication No.20020023277 A1 to Stuhlmann, H et al discloses the identification andisolation of the vasculogenesis-related gene Vezf1 in mice, and methodsfor selection of endothelial cells and precursors based on Vezf1expression. Also disclosed are methods for modulating angiogenesis, anddiagnosis and treatment of vascular disease and neoplasm in a subject,the methods employing detection, measurement and modification of levelsof Vezf1 in tissues. However, the transgenic ES cell experimentsdescribed were restricted to mouse embryoid body cells only, and neitherhuman nor any other primate embryo cells were used. Furthermore,selection, according to the disclosure, is on the basis of Vezf1expression, thus failing to overcome the abovementioned limitations ofaggregation and immunofluorescent sorting.

U.S. Patent Application Publication No. 20020039724 A1 to Carpenter, M Kdiscloses methods for differentiation and selection of human embryonicneural progenitor cells, and therapeutic, diagnostic and investigativeuses thereof. The disclosed human neural progenitor cells, forreconstitutive therapy of, for example, neural degenerative disease, arealso derived from human embryoid bodies, and are selected and isolatedaccording to expression and detection of neural cell specific markers,NCAM and A2B5. Similarly, International Patent Application WO 0181549 A3to Rambhatla L and Carpenter M K discloses methods for treating embryoidbodies with n-butyrate for induction of differentiation into hepatocytelineage cells. No mention is made of non-aggregated hES origins, orsimplified methods of progenitor isolation in either application.

Recently, Benevenisty (International Patent Application WO 0210347 A2 toBenvenisty) disclosed methods for “directed differentiation” of humanembryonic stem cells by treating aggregated, embryoid body-derived cellswith exogenous factors, enriching the cultures for a specific lineagecell type. The factors used were known effectors of differentiation,such as retinoic acid, neuronal growth factor, epidermal growth factor,fibroblast growth factor, etc., and differentiation was determined by denovo gene expression, and the appearance of tissue lineage-specific cellsurface markers.

U.S. Pat. Application Publication No. 20010041668 A1, to Baron, M et al,discloses the use of extraembryonic, morphogenic gene products such asHedgehog, TNF and WNT for modulation of hematopoiesis and vasculargrowth from mammalian adult and embryonic mesodermal-derived stem cells.Manipulation of the levels of these extra-embryonic gene products in thestem cell environment, via external application, or genetic engineering,for example, is disclosed for either enriching or diminishing thehematopoietic and/or vascular potential of stem cells for treatment anddiagnosis of diseases involving blood abnormalities,hypervascularization, neovascularization and revascularization oftissues. However, although treatment of human embryonic tissues isproposed, no examples using human adult or embryonic cells arepresented, and no methods for culture or selection of non-aggregatedembryonic stem cells, designed to overcome the abovementionedlimitations, are disclosed.

Thus, there exists a need for a simplified and inexpensive method forthe in-vitro identification, isolation and culture of human vasculogenicprogenitor cells. Such a method and the progenitor cells isolatedthereby can be used for in-vitro vascular engineering, treatment ofcongenital and acquired vascular and hematological abnormalities, forevaluation and development of drugs affecting vasculo- and angiogenicprocesses, and for further investigation into tissue differentiation anddevelopment.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preparing vasculogenic progenitor cells from undifferentiatedES cells, the method effected by culturing individual undifferentiatedES cells in a manner suitable for inducing differentiation of theundifferentiated ES cells into vasculogenic progenitor cells, therebyobtaining a mixed population of cells; and isolating cells smaller than50 μm from said mixed population of cells, said cells smaller than 50 μmbeing vasculogenic progenitor cells.

According to another aspect of the present invention there is provided amethod of preparing epithelial progenitor cells from undifferentiated EScells, the method effected by culturing individual undifferentiated EScells in a manner suitable for inducing differentiation of theundifferentiated ES cells into vasculogenic progenitor cells therebyobtaining a mixed population of cells; and isolating cells larger than50 μm from said mixed population of cells, said cells larger than 50 μmbeing epithelial progenitor cells.

According to yet another aspect of the present invention there isprovided a method of preparing somatic cells from a population ofvasculogenic progenitor cells, the method effected by obtaining apopulation of vasculogenic progenitor cells; and culturing thepopulation of vasculogenic progenitor cells in the presence of at leastone growth factor suitable for inducing somatic cell differentiation.

According to yet another aspect of the present invention there isprovided a method of generating vascular smooth muscle cells fromvasculogenic progenitor cells. The method is effected by culturing thevasculogenic progenitor cells in a differentiating medium including aserum volume concentration higher than 5% for a time period sufficientfor inducing differentiation of the vasculogenic progenitor cells intovascular smooth muscle cells.

According to still another aspect of the present invention there isprovided a method of generating endothelial cells from vasculogenicprogenitor cells. The method is effected by culturing the vasculogenicprogenitor cells in a differentiating medium including a serum volumeconcentration lower than 5% by volume for a time period sufficient forinducing differentiation of the vasculogenic progenitor cells intoendothelial cells.

According to a further aspect of the present invention there is provideda method of enhancing differentiation, maturation and/or functionalityof vasculogenic cells. The method is effected by exposing thevasculogenic cells to a shear force of at least 1 dyne/cm² for a timeperiod sufficient to enhance differentiation, maturation and/orfunctionality of the vasculogenic cells

According to still another aspect of the present invention there isprovided a method of preparing vascular tissue, the method is effectedby obtaining a population of vasculogenic progenitor cells; andculturing the population of vasculogenic progenitor cells in thepresence of at least one vasculogenic and/or angiogenic growth factor,under conditions suitable for inducing vascular tissue differentiation.

According to further features in preferred embodiments of the inventiondescribed below the population of vasculogenic progenitor cells iscultured in a semi-solid, vascularization-promoting medium.

According to yet further features in preferred embodiments of theinvention described below the population of vasculogenic progenitor iscultured on a 3-dimensional scaffold.

According to still further features in preferred embodiments of theinvention described below the vasculogenic and/or angiogenic factor isselected from the group consisting of vascular endothelial growth factor(VEGF), angiopoietin (Ang), platelet derived growth factor (PDGF),ephrin (Eph), fibroblast growth factor (FGF), tumor growth factor (TGF)and placental growth factor (PIGF).

According to an additional aspect of the present invention there isprovided a method of determining an effect of a factor on vasculardevelopment, growth and/or modification, the method effected byobtaining a population of vasculogenic progenitor cells; exposing thepopulation of vasculogenic progenitor cells to the factor; anddetermining an effect of the factor on the population of vasculogenicprogenitor cells to thereby determine the effect thereof on vasculardevelopment.

According to further features in preferred embodiments of the inventiondescribed below the factor is a substance and/or an environmentalfactor.

According to yet further features in preferred embodiments of theinvention described below the factor is a putative angiogenesis and/orvasculogenesis downregulator, whereas the method further includesculturing the population of vasculogenic progenitor cells underconditions suitable for promoting angiogenesis and/or vasculogenesis.

According to still further features in preferred embodiments of theinvention described below the factor is a putative angiogenesis and/orvasculogenesis upregulator, whereas the method further includesculturing the population of vasculogenic progenitor cells underconditions limiting angiogenesis and/or vasculogenesis.

According to a further aspect of the present invention there is provideda method of relieving or preventing a vascular disease or condition in amammalian subject, the method effected by obtaining a population ofvasculogenic progenitor cells; and administering the vasculogenicprogenitor cells into the subject under conditions suitable forstimulating differentiation of the vasculogenic progenitor cells intoendothelial and smooth muscle cells.

According to further features in preferred embodiments of the inventiondescribed below the vascular disease or condition is selected from agroup consisting of congenital vascular disorders, acquired vasculardisorders and ischemia/reperfusion injury.

According to yet a further aspect of the present invention there isprovided a method of vascularizing a mammalian tissue, the methodeffected by obtaining a population of vasculogenic progenitor cellscontacting the vasculogenic progenitor cells with the mammalian tissueunder conditions suitable for stimulating differentiation of thevasculogenic progenitor cells into endothelial and smooth muscle cells.

According to further features in preferred embodiments of the inventiondescribed below the mammalian tissue is an engineered, non-vasculartissue in need of vascularization and/or an embryonic tissue.

According to further features in preferred embodiments of the inventiondescribed below contacting the vasculogenic progenitor cells with themammalian tissue is performed in vitro or in vivo.

According to still a further aspect of the present invention there isprovided a method of relieving or preventing a hematological disease orcondition in a mammalian subject, the method effected by obtaining apopulation of vasculogenic progenitor cells; and administering thevasculogenic progenitor cells into the subject under conditions suitablefor stimulating differentiation of the vasculogenic progenitor cellsinto endothelial and blood cells.

According to further features in preferred embodiments of the inventiondescribed below the hematological disease or condition is selected froma group consisting of congenital blood disorders, acquired blooddisorders, clotting disorders and neoplastic disease.

According to further features in preferred embodiments of the inventiondescribed below obtaining the population of vasculogenic cells iseffected by culturing individual undifferentiated ES cells in a mannersuitable for inducing differentiation of the undifferentiated ES cellsinto vasculogenic progenitor cells thereby obtaining a mixed populationof cells and isolating cells smaller than 50 μm from said mixedpopulation of cells.

According to still an additional aspect of the present invention thereis provided a composition of matter comprising a substrate and apopulation of vasculogenic progenitor cells, wherein said vasculogenicprogenitor cells are prepared from undifferentiated ES cells by a methodeffected by the steps: culturing individual undifferentiated ES cells ina manner suitable for inducing differentiation of the undifferentiatedES cells into vasculogenic progenitor cells thereby obtaining a mixedpopulation of cells and isolating cells smaller than 50 μm from saidmixed population of cells, said cells smaller than 50 μm beingvasculogenic progenitor cells.

According to further features in preferred embodiments of the inventiondescribed below the substrate is selected from the group consisting ofmatrigel, collagen gel, and polymeric scaffold.

According to still further features in preferred embodiments of theinvention described below the vasculogenic progenitor cells is contactedwith the substrate in a manner so as to induce vascular developmentwithin the substrate.

According to further features in preferred embodiments of the inventiondescribed below the hematological disease or condition is selected froma group consisting of congenital blood disorders, acquired blooddisorders, clotting disorders and neoplastic disease.

According to yet further features in preferred embodiments of theinvention described below culturing the individual undifferentiated EScells is effected by subjecting the undifferentiated ES cells to atleast one condition selected from a group consisting of avoidingaggregation of ES cells, growth on collagen, cell seeding concentrationbetween 2×10⁴ and 1×10⁵ cells/cm² and presence of differentiationmedium.

According to still further features in preferred embodiments of theinvention described below the undifferentiated ES cells are human EScells.

According to an additional aspect of the present invention there isprovided a method of preparing endothelial cells from vascular tissue,the method effected by subjecting the vascular tissue to conditionsdesigned for dissociating cells from the vascular tissue, therebyobtaining a mixed population of dissociated cells and isolating cellssmaller than 50 μm from said mixed population of cells.

According to a further aspect of the present invention there is provideda method of preparing epithelial cells from vascular tissue, the methodeffected by subjecting the vascular tissue to conditions designed fordissociating cells from the vascular tissue, thereby obtaining a mixedpopulation of dissociated cells, thereby obtaining a mixed population ofindividual cells; and isolating cells larger than 50 μm from said mixedpopulation of cells.

According to further features in preferred embodiments of the inventiondescribed below the vascular tissue is human vascular tissue

According to yet further features in preferred embodiments of theinvention described below the cells smaller or larger than 50 μm areisolated via filtration, morphometry and/or densitometry.

According to still further features in preferred embodiments of theinvention described below the filtration is effected via a filter havinga pore size smaller than 50 μm.

According to yet an additional aspect of the present invention there isprovided a cell culture comprising a population of vasculogenicprogenitor cells being sustainable in a proliferative state for at least14 days and being capable of differentiation into smooth muscle,endothelial and/or hematopoietic cells upon exposure to at least onegrowth factor selected from the group consisting of vascular endothelialgrowth factor (VEGF), angiopoietin (Ang), platelet derived growth factor(PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growthfactor (TGF), placental growth factor (PIGF), cytokines, erythropoietin,thrombopoietin, transferrin, insulin, stem cell factor (SCF),Granulocyte colony-stimulating factor (G-CSF) and Granulocyte-macrophagecolony stimulating factor (GM-CSF).

According to further features in preferred embodiments of the inventiondescribed below the population of vasculogenic progenitor cells iscapable of expressing at least one exogenous polypeptide selected fromthe group consisting of cell-surface markers, cell-surface antigens,angiogenic factors, vasculogenic factors and hematopoietic factors.

According to still further features in preferred embodiments of theinvention described below the exogenous polypeptide is expressed in aninducible manner.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-I provide an outline for, and micrographs demonstrating theculture-based selection for human ES cell-derived vasculogenicprogenitor cells.

FIG. 1A illustrates the outline of the differentiation-selectionprocedure. FIG. 1B is a series of micrographs demonstrating thedivergent morphology of the cells following 6 days culture on collagen:note the large, flat fiber-bearing cells (arrows) and the smaller,flattened cells with large nuclei (arrowheads). FIG. 1C is a series ofgraphs demonstrating a FACS analysis of endothelial cell surface markersin the filtered, isolated vasculogenic progenitors cells. Filtered cellswere exposed to primary antibodies to VE-cadhedrin (VE-cad), VEGFR2(VEGFR2), and to fluorescent labeled anti-IgG, or to the second antibodyalone (IgG-FITC). Note the high proportion of cells (78%) expressingVE-cad. FIGS. 1D-E are photographs demonstrating the indirectimmunomorphological analysis of VE-cad expression on filtered, isolatedvasculogenic progenitors cells. Immunofluorescent staining of fixed andplated 12 hour cultures of the filtered cells demonstrate stronglocalization of VE-cad, at cell-cell adherent junctions, visible athigher magnification (FIG. 1E). FIG. 1F is a photograph of EtBr stainedgels demonstrating the expression of endothelial and hematopoieticmarkers in the isolated vasculogenic progenitors cells. Expression ofthe CD31 and Tie2 endothelial markers and the Tal1, GATA2 and AC 133early vasculogenic progenitor markers was compared in total RNA from thesmaller, flat filtered cells (Filtrated), and the undifferentiated humanembryonic stem (hES) cells by RT-PCR. The housekeeping marker GAPDHserves as an internal standard of amplification. Note the prominent,endothelial, smooth muscle and hematopoietic (ESH)-specific expressionof the CD31, Tie2, Tal1 and GATA2 markers. FIG. 1G is a fluorescentmicrograph of the larger, flat cells retained by filtration,demonstrating the presence of the epitheliod phenotype smooth musclecell marker α-sma not detected in the smaller, human vasculogenicprogenitor cells. FIG. 1H is a photograph of EtBr stained gelsdemonstrating the expression of epitheliod markers in the isolated,larger retained cells. Expression of the Calponin, Caldesmon, smoothmuscle actin (SMA) and SM-MHC markers was compared in total RNA from thelarger, flat retained cells (Retained), and the smaller, humanvasculogenic progenitor (Filtrated) cells by RT-PCR. The housekeepingmarker GAPDH serves as an internal standard of amplification. Note theabsence of expression of all of the smooth muscle cell markers in thehuman vasculogenic progenitor cells, and their prominent expression inthe Retained cells. FIG. 1I is a micrograph of BrdU incorporation inboth smaller, filtered (left panel), and larger, retained cells (rightpanel), demonstrating active proliferation of the smaller, humanvasculogenic progenitor cells. Note the active uptake of BrdU in thesmaller, darkened cell nuclei, contrasted with the minimal incorporationin the larger, non-proliferating retained cells (arrow). Bar equals 100μm.

FIGS. 2A-M are microscopic, immunofluorescence and RT-PCR studies ofcultured common human vasculogenic progenitor cells, demonstratingspecific growth factor-mediated induction of endothelial or smoothmuscle cell characteristics. FIG. 2A is a photograph of EtBr stainedgels demonstrating the expression of smooth muscle cell markers incommon human vasculogenic progenitor cells recultured on type IVcollagen at 2.5×10⁴ cells/cm² for 10-12 days with 10 ng/ml hPDGF-BB (Rand D Systems, Inc., Minneapolis, Minn., USA). Expression of the smoothmuscle cell markers Caldesmon, smooth muscle actin (SMA), Calponin,SM22α and SM-MHC markers was compared in total RNA from the growthfactor-treated cells (v-SMC), and the untreated human vasculogenicprogenitor (ESH progenitor) cells by RT-PCR. The housekeeping markerGAPDH serves as an internal standard of amplification. Note the absenceof expression of all of the smooth muscle cell markers in the ESH cells,and their prominent expression in the HPDGF-BB treated cells. FIGS. 2B-Eare photomicrographs of immunofluorescent detection of smooth musclecell markers expressed in the human platelet-derived growth factor(hPDGF)-BB-treated human vasculogenic progenitor cells. Fixedpreparations of treated cells were stained with primary antibodies to: aSMA (FIG. 2B); smoothelin (FIG. 2C); SM-MHC (FIG. 2D), and Calponin(FIG. 2E), immunodetected with fluorescent second antibodies andvisualized via fluorescent microscopy. Note the staining of bothepitheliod and spindle-shaped cell types in the growth factor-treatedcultures. FIGS. 2F-H are photomicrographs showing the detection ofendothelial cell markers expressed in human vasculogenic progenitorcells recultured on type IV collagen at 2.5×10⁴ cells/cm² for 10-12 dayswith 50 ng/ml hVEGF₁₆₅ (R and D Systems, Inc. Minneapolis, Minn., USA).Growth factor-treated cells were fixed and immune-detected as describedhereinabove with anti-VEcad (FIG. 2F), or anti-von Willebrand Factor(vWF)(FIG. 2G) antibodies. Note the localization of anti vWF staining inthe Weibel-Palade bodies (FIG. 2G). Uptake of Dill-labeled ac-LDL (10μg/ml, 4 hours, 37° C.)(FIG. 2 h) was also detected (FIG. 2H). FIGS. 2Iand 2J are micrographs of BrdU incorporation in both platelet derived(PDGF) (FIG. 2J) and vascular endothelium (VEGF) (FIG. 2I) growthfactor-treated human vasculogenic progenitor cells, demonstrating activeproliferation (staining) of the endothelial-type cells. Note theappearance of stress fibers (FIG. 2I, arrow), and the active uptake ofBrdU in the darkened cell nuclei of the VEGF-treated cells (FIG. 2I),contrasted with the reduced incorporation of the larger hPDGF-BB treatedcells (FIG. 2J). FIGS. 2K-2M are micrographs of hematopoietic coloniesformed from human vasculogenic progenitor cells. ESH cells wereselected, and cultured in a semisolid medium supplemented with cytokinesto promote hematopoietic differentiation. Note the characteristicappearance of hematopoietic colonies (CFU) detected after 12 daysincubation.

FIGS. 3A-G are photomicrographs (FIGS. 3A-D), and electron micrographs(FIGS. 3E-G) showing vascular structure formation ingrowth-factor-treated human vasculogenic progenitor (ESH) cells.Aggregated (24 hours in differentiation medium supplemented with 50ng/ml hVEGF₁₆₅ and 10 ng/ml hPDGF-BB) ESH cells seeded onto type Icollagen (FIG. 3A) or in matrigel (FIG. 3B) demonstrated vascularformation after 7 days growth (Note sprouting and tubular structures inboth histology sections). Toluidine blue-stained sections of the samepreparation revealed endothelial cell penetration and formation of avascular-network structure in the matrigel (FIG. 3C) and, with highermagnification, a white blood cell formed within a vessel (arrow, FIG.3D). Bar equals 100 μm (FIGS. 3A-C) (FIG. 3D). FIGS. 3E-G are electronmicrographs of vessel formation in Matrigel, showing well-formedWeibel-Palade bodies (FIG. 3E, ×6,000 magnification, inset, ×12,000),typical of endothelial cells. FIG. 3F clearly demonstrates the presenceof a darkly staining (due to Hemoglobin) blood cell (BC) in the centerof a vessel formed by elongated endothelial cells (EC) within thematrigel (M) (×5,000 magnification). FIG. 3G demonstrates typicalarrangement of endothelial cells (N-nucleus) within the matrigel (M),containing a clearly discernible lumen (Lu), characteristic lipoproteincapsules (Li), Weibel-Palade bodies (WP) and glycogen (G) (FIG. 3G,×5,000 magnification).

FIGS. 4A-B are photomicrographs of histology sections depicting the invitro vascularization of 3-D alginate scaffolds by human vasculogenicprogenitor (ESH) cells. ESH aggregates were seeded on If120 50 μlalginate scaffolds in vitro in differentiation medium supplemented with50 ng/ml hVEGF₁₆₅ and 10 ng/ml hpDGF-BB, and incubated for 14 days. FIG.4A shows vessel formation around two representative scaffold pores.Higher magnification (FIG. 4B) reveals typical vascular wall structureof elongated flat endothelial cells with an adjacent layer of smoothmuscle cells. Bar equals 100 μm.

FIGS. 5A-B are two series of photomicrographs demonstrating thesensitivity of ESH-derived vascular tissue to inhibitors ofangiogenesis. ESH aggregates were seeded on matrigel and incubated for 7days in differentiation medium supplemented with 50 ng/ml hVEGF₁₆₅ and10 ng/ml hPDGF-BB alone (FIG. 5A) or with the addition of 50 μg/mlangiogenesis inhibiting anti VE-cad monoclonal antibody (clone BV6,CHEMICON INTNL, Inc. Temecula Calif., USA)(FIG. 5B). Notethe lack ofcellular projections and absence of tube and network structures in theanti VE-cad treated cultures (FIG. 5B). Bar—100 μm.

FIGS. 6A-F illustrate culture based enrichment of vascular progenitorcells derived from hES cells. FIG. 6A-presents an outline of theenrichment procedure. FIG. 6B is an inverted light microscopy image of 6day old hES cell-aggregates-cultured on type IV collagen. This imageshows undifferentiated hES cells (arrows) and different types ofdifferentiated cells. FIG. 6C is an inverted light microscopy image of 6day old single-cell-suspension-cultures illustrating two cell types: bigflat cells with fiber arrangement (arrow) and smaller flat cells withlarge nuclei. FIG. 6D is a FACS analysis of the filtrated cells forVE-cad, CD31 and VEGFR2. FIG. 6E is an indirect immunofluorescenceanalysis showing expression of: (i) punctate surface CD34 (as previouslyreported for human v-SMCs progenitors), (ii) nuclei Gata2 and (iii)Tal1. FIG. 6F demonstrates cell proliferation of the smaller and largerprogenitors via: (i and ii) BrdU incorporation (present in the smallprogenitor cells and not the larger cells (arrow) and (iii) nucleic Ki67expression which is present in 66±2% of the filtrated cells. Nucleistained with Dapi (1:1000). Bar —100 μm.

FIGS. 7A-D illustrate lineage differentiation in progenitor cells. FIG.7A-filtrate cells recultured with 50 ng/ml hVEGF165 for 10-12 days wereexamined for Dil-Ac-LDL incorporation (cells in bright light on the leftand in fluorescence illumination on the right). FIG. 7B-individualsegregated cells were examined for: (i) Dil-Ac-LDL metabolism (ii)perinuclear vWF, (iii) both (i) and (ii). FIG. 7C-filtrate cellsrecultured with 10 ng/ml hPDGF-BB for 10-12 days exhibited up-regulationof SMA expression in spindle-like shape cells. FIG. 7D-RT-PCR analysisrevealed an up-regulation in additional v-SMC markers. Nuclei stainedwith Dapi (1:11000). Bar—100 μm

FIGS. 8Ai-Biii illustrate clonal analysis of VE-cad+ cells. FIG. 8A(i)illustrates a typical 8-day-old colony formed from a single VE-cad+cell. Two distinct cell shapes were observed: an endothelial cell likemorphology [FIG. 8A(ii)] and spindle-like morphology (arrows) resemblingv-SMC [FIG. 8A(iii)]. Spindle shaped cells expressed SMA [FIG. 8B(i)]and calponin [FIG. 8B(ii)]. FIG. 8B(iii) illustrates Ac-LDL metabolismin colony supplemented with VEGF. Nuclei stained with Dapi. Bar—100 μm.

FIGS. 9Ai-Ciii are photomicrographs of histology sections depictingblood and blood vessels formed within alginate scaffolds seeded withhuman ES vasculogenic progenitor cells and transplanted subcutaneouslyin SCID mice. FIG. 9A illustrates immature blood vessels having a thinlayer of endothelial cells formed within non-seeded (control) scaffolds.FIG. 9B illustrates thick blood vessels formed within cell seededscaffolds. FIG. 9C illustrates blood vessels of human origin formedwithin cell-seeded scaffolds and identified by anti-human SMA staining.Arrows indicate on mouse blood-flow within the human vasculature.Bar—1100 μm.

FIG. 10 is a schematic illustration of the flow chamber used forevaluating the effect of shear stress on vasculogenic cells.

FIGS. 11A-B are photomicrographs illustrating vasculogenic smooth musclecells (v-SMC) derived from hES vasculogenic progenitor cells followingexposure to flow-induced shear stress. The v-SMC cells were stained foraSMA (red), phalloidin (green) and nuclei in To-pro 3 (blue).

FIG. 12 illustrates RT-PCR analyses of differentiating cultures ofhES-derived vasculogenic progenitor cells. The cells were cultured inlow serum (2%) or high serum (10%) differentiating media supplementedwith growth factors (VEGF, Ang2 or IGF). The analyses were performedusing genetic markers of endothelial cells (ECs), vascular smooth musclecells (v-SMCs) and growth factors; − and + indicate negative (withouttemplate) and positive controls, respectively.

FIG. 13 illustrates real-time RT-PCR analyses of differentiatingcultures of hES-derived vasculogenic progenitor cells. The cells werecultured in low serum (2% v/v) or high serum (10% v/v) differentiatingmedia supplemented with VEGF. The analyses were performed using v-SMCsmarkers (SM-MHC and α-SMA).

FIG. 14 illustrates real-time RT-PCR analyses of differentiatingcultures of hES-derived vasculogenic progenitor cells. The cells werecultured in low serum (2% v/v) or high serum (10% v/v) differentiatingmedia supplemented with VEGF. The analyses were performed using ECmarkers (Tier2 and CD31).

FIGS. 15A-B are photomicrographs illustrating sprouting andvasculature-like organization of differentiated cells derived from hESvasculogenic progenitor cells and cultured in high serum differentiatingmedium (10% v/v; FIG. 15A) and in low serum differentiating medium (2%v/v; FIG. 15B).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel methods which can be used for simpleand inexpensive preparation of vasculogenic progenitor cells, and cellcultures and compositions thereof prepared from, for example, human stemcells. Specifically, the present invention can be used for isolatingvasculogenic progenitor cells from stem cells, and for in vitro growthand differentiation of the isolated vasculogenic progenitor cells foruse in, for example, tissue engineering, angiogenesis research,therapeutic and diagnostic applications.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Recent research studies have illustrated that embryonic cells canpotentially serves as a source for pluripotent cells. Such cells areuseful in human therapy since they posses the capacity to differentiateinto a plurality of cell types (R. A. Pedersen, Sci. Am. 1999; 280:68).Early work on embryonic stem cells was done using inbred mouse strainsas a model. Compared with mouse ES cells, monkey and human pluripotentcells have proven to be much more fragile, and do not respond to thesame culture conditions and manipulations.

Recently, human embryonic stem cells (hES) and germ-line (hEG) cellshave been isolated and maintained in culture. Both human embryonic hESand hEG cells have the long-sought characteristics of human pluripotentstem cells, they are capable of ongoing proliferation in vitro withoutdifferentiating, they retain a normal karyotype, and they retain thecapacity to differentiate to produce all adult cell types. However,spontaneous somatic differentiation of hES and hEG cells in cultureproceeds without any consistent pattern of structural organization,generating multicellular aggregates of cell populations with a highlyheterogeneous mixture of phenotypes, representing a spectrum ofdifferent cell lineages (Reubinoff, B E, et al Nat Biotech 2001;19:1134).

Prior art studies describe various methods suitable for isolation ofprogenitor cells of specific cell type lineages from ES cells, however,such methods are typically extremely complex and costly. Initially,human embryonic stem cells are either grown on a mammalian stromal celllayer (see, for example, U.S. Pat. No. 6,280,718 to Kaufman, D S andThomson, J A), in a live host as a teratoma (Thomson J A et al Science1998; 282:1145-47) or aggregated in suspension into a multicellularstructure known as the embryoid body (EB) (see, for example,International Pat. Application No. WO0070021 A3 to Itskovitz-Eldor J andBenvenisty N; and International Pat. Application WO0210347 A2 toBenvenisty N), and exposed to differentiation factors, typicallyproducing a mixed population of cell types and lineages. Isolation ofprogenitor cells of specific lineages is then accomplished on the basisof immunodetection of lineage-specific markers, and separation of celllineages by fluorescent or magnetic sorting (see, for example,International Pat. Application No. WO0181549 A3 to Rambhatla L andCarpenter, M K; U.S. Pat. No. 6,280,718 to Kaufman, D S and Thomson, JA; International Pat. Application No. WO0129206 A1 to Cibelli, J et al;and International Pat. Application No. WO 0168815 A1 to Pera, M F andBen-Hur T). All of the abovementioned methods suffer from similardisadvantages: initial ES differentiation into progenitor cells involvesmany complex manipulations and interactions with the stromal cell layer,live host tissues or other EB cells. Furthermore, selection according toexpression or display of cell surface markers is inefficient, requiringeven more extensive manipulation, incurring great expense for reagentsand detection equipment, and endangering the vitality and sterility ofthe progenitor cells.

While reducing the present invention to practice the present inventorshave uncovered that vasculogenic progenitor cells may be prepared simplyand inexpensively from embryonic stem cells by preventing aggregation,culturing on type IV collagen with specific endothelial differentiationfactors and employing simple and efficient size selection methods. Thevasculogenic progenitor cells prepared by the present invention areadvantageous in that they can be further expanded in culture, can beinduced to differentiate into endothelial, mural and hematopoietictissue in vitro, form both small and large vascular structures whenseeded on appropriate substrate, may be genetically manipulated and aresuitable for tissue engineering, diagnostic and research purposes.

Thus, according to one aspect of the present invention there is provideda method of preparing vasculogenic progenitor cells fromundifferentiated ES cells, such as human ES cells. The method, accordingto this aspect of the present invention, is effected by culturingindividual undifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells, thereby obtaining a mixed population of cells, andisolating cells smaller than 50 μm from the mixed population of cells.Cell isolated in this manner are vasculogenic progenitor cells as isclearly illustrated in the Examples section hereinunder.

As used herein, the phrase “vasculogenic progenitor cells” refers to apopulation of cells that can generate progeny that are endothelial orsmooth muscle precursors (such as angioblasts) or mature endothelial orsmooth muscle cells, or hematopoietic precursor (such as erythroidcolony forming units and megakaryocytes) or mature blood cells (such aserythrocytes and leukocytes). Typically, vasculogenic progenitor cellsexpress some of the phenotypic markers that are characteristic of theendothelial, smooth muscle and hematopoietic lineages. Typically, theydo not produce progeny of other embryonic germ layers when cultured bythemselves in vitro, unless dedifferentiated or reprogrammed. It will beappreciated that it is not implied that each of the cells within thepopulation have the capacity of forming more than one type of progeny,although individual cells that are multipotent vasculogenic progenitorcells may be present.

As used herein the, the terms “totipotent”, “pluripotent” and“multipotent” refer to cells having decreasing degrees of developmentalplasticity. Totipotent cells are capable of developing into all celltypes or complete organisms (e.g. blastomeres), pluripotent cellscapable of differentiating into all cell types (e.g. ES cells) andmultipotent cells are capable of differentiating into cells of specificlineages only (e.g. vasculogenic progenitor cells).

As used herein, the term “endothelial progenitor cell” or “endothelialprecursor cell” refers to a cell that can generate mature endothelialcells. These cells may or may not have the capacity to generatehematopoietic or smooth muscle cells.

As used herein, the term “epithelial progenitor cell” or “epithelialprecursor cell” refers to a cell that can generate mature smooth musclecells.

As used herein, the term “hematopoietic progenitor cell” or“hematopoietic precursor cell” refers to a cell that can generate matureblood cells.

Embryonic stem cells are described as “undifferentiated” when asubstantial portion of stem cells and their derivatives in thepopulation display morphological characteristics of undifferentiatedcells, clearly distinguishing them from differentiated cells ofembryonic or adult origin. Undifferentiated ES cells are easilyrecognized by those skilled in the art, and typically appear in amicroscopic view as cells with high nuclear/cytoplasm ratios andprominent nucleoli. Similarly, undifferentiated cells can bedistinguished from differentiated cells by the absence of lineagespecific markers such as vascular endothelial growth factor receptor 2(VEGFR2), vascular endothelial cadherin (VE-cad) or platelet-endothelialcell adhesion molecule-1 (PECAM-1).

As used herein, the term “differentiated cell” refers to a cell that hasprogressed down a developmental pathway. Thus, pluripotent embryonicstem cells can differentiate to lineage-restricted precursor cells, suchas neural progenitor, hepatocyte progenitor or hematopoietic cells,which are pluripotent for neural cells, hepatocytes and blood celltypes, respectively; and the endothelial, smooth muscle and blood celltypes listed above. These in turn may be differentiated further intoother types of precursors further down the pathways, or to an end-stagedifferentiated cell, which is characteristic of a specific tissue type,and may or may not retain the capacity to proliferate further. Vascularendothelium, mural smooth muscle and erythrocytes are examples ofterminally differentiated cells.

As mentioned hereinabove, individual undifferentiated ES cells arecultured in a manner suitable for inducing differentiation intovasculogenic progenitor cells. The undifferentiated ES cells utilized bythe method of the present invention can be mammalian embryonic stemcells obtained from fresh or cryopreserved embryonic cell masses, cellsfrom in-vitro-fertilized embryonic cell masses and/or cultured ES celllines. The ES cells may be of human or non-human origin.

As is clearly demonstrated in the Examples section hereinbelow, themethods and compositions of the present invention are suitable for usewith human embryonic stem cells. Since establishment of methods formanipulation and control of human embryonic stem cell differentiation isa primary goal of current medical and scientific effort, in a preferredembodiment of the present invention, the undifferentiated ES cells arehuman ES cells. Preferably, the ES cells are unaggregated cells, asdescribed in detail in the Examples section hereinbelow.

According to another preferred embodiment of the present invention,differentiation of the individual undifferentiated ES cells is effectedby culturing such cells on plates coated with an adhesive substrate suchas type IV collagen, laminin or gelatin to prevent aggregation of the EScells, seeding the cells at a concentration between 2×10⁴ and 1×10⁵cells/cm², and providing differentiation medium. In a most preferredembodiment, individual undifferentiated ES cells are grown on type IVcollagen-coated plates (available from, for example, Cell Cultureware, BD-Falcon, Boston, Mass.). See Examples section for further descriptionof conditions for differentiation of ES cells.

One important feature of the present methodology is the cell seedingstep. While reducing the present invention to practice, it was observedthat a 3-dimensional embryoid body structure was not required, as hadbeen previously contended, for mesodermal differentiation of humanembryonic stem cells. Undifferentiated hES cells removed from theirfeeder layer and plated as single cells on type IV collagen withdifferentiation medium exhibited expression of indicators of endothelialdifferentiation (FIGS. 1A-G). Cell seeding concentration dramaticallyaffected the efficiency of the present methodology: cells seededaccording to prior art studies with mouse ES (Yamashita, J et al Nature2000; 408:92) were not viable; such high concentrations (1.0−1.5×10⁵cells/cm²) of hES cells resulted in a heterogeneous population. Lowercell seeding concentrations (5×10⁴-1×10⁵ cells/cm²) produced a definedpopulation of cells, including a majority of small, flatendothelial-like cells and fewer large, smooth-muscle-like cells (FIG.1B).

The Examples section which follows provides further description ofmethods of culturing “individual undifferentiated ES cells” undernon-aggregating conditions.

As used herein, the term “differentiation medium” refers to a suitablemedium capable of supporting growth and differentiation of the ES cells.Examples of suitable differentiation media which can be used with thepresent invention include a variety of growth media prepared with a baseof alpha MEM medium (Life Technologies Inc., Rockville, Md., USA) orDulbecco's minimal essential medium (DMEM) supplemented with 10% FBS(HyClone, Logan, Utah, USA) and 0.1 mM β-mercapoethanol (LifeTechnologies Inc., Rockville, Md., USA).

As is mentioned hereinabove it was observed that culturing of theundifferentiated ES cells as detailed hereinabove produces a definedpopulation of cells, including a majority of small, flatendothelial-like cells and fewer large, smooth-muscle-like cells (FIG.1B).

While previous techniques for selection of specific lineage progenitorshave depended on immunodetection of indicators of differentiation andspecific cell lineages and fluorescent or magnetic cell sorting (see,for example, International Patent Application WO 0210347 A2 toBenvenisty, U.S. Pat. No. 6,280,718 to Kaufman, D S and Thomson, J A;International Pat. Application No. WO0129206 A1 to Cibelli, J et al),these methods are cumbersome and costly. The observed morphologicalfeatures of the mixed population of cells generated according to theteachings of the present invention enabled a simple and rapid isolationof vasculogenic progenitor cells therefrom. As is illustrated in theExamples section which follows, selection of cells smaller than 50 μm,enables rapid and efficient isolation of vasculogenic progenitor cellsfrom the mixed population of cells (FIGS. 1C-F).

Thus, the present methodology employs a step of size/morphologyselection following differentiation. Such size/morphology selection canbe effected using various filtration, morphometry and/or densitometryapproaches as is further described below.

Methods of filtration are well known in the art, such as the passagethrough a mesh, sieve, filter and the like. Filters can comprise afibrous matrix or porous material. Such filters may be one of severalcommercially available filters including but not limited to cell culturefilters from Pall Life Sciences (Ann-Arbor Mich., USA) or BD-Falcon(Boston, Mass., USA). A preferred filter is a nylon mesh filter having apore size of 40 μm (Cell Cultureware, BD-Falcon, Boston, Mass.),allowing the smaller, endothelial-like cells to pass and the larger,smooth-muscle like cells to be excluded.

“Morphometry” refers to the measurement of external form, and can employmethods including but not limited to 2- and 3-D image analysis. Advancedimaging analysis software suitable for identification and isolation ofcells smaller than 50 μm is commercially available to one skilled in theart [see, for example, Metamorph Software (Universal Imaging Corp.,Downing Pa., USA), Imagic-5 (Image Science Software, Berlin, Germany)and Stereologer (Systems Planning and Analysis, Inc., Alexandria, Va.,USA)] and can be combined with well known light microscopy and flowsorting techniques for selection of objects of desired externalcharacteristics (e.g. size) (for suitable apparatus see, for example,U.S. Pat. No. 6,249,341 to Basiji et al).

“Densitometry” refers to measurement of the optical or physical densityof an object. Since the smaller, endothelial-like cells have a uniqueand characteristic distribution of cell components, densitometricmeasurements may be used to characterize and provide criteria forseparation and isolation of cells. Devices suitable for densitometricisolation of endothelial-like cells are, for example, the MECOS-C1 bloodcell densitometry analyzer (MECOS Co., Moscow, Russia). Cells may alsobe separated by sedimentation through a preparative density gradientsuch as FICOLL™ or PERCOLL™ (Amersham Biosciences, Inc. Piscataway, N.J.USA) (for exhaustive review of densitometric fractionation techniques,see Pertoft, H J Biochem Biophys Methods 2000; 44:1-30). Thus, thepresent invention provides an easy and rapid approach to progenitor cellgeneration and isolation. Previous methods of isolating such progenitorcells have produced progenitor populations which lack desirableproliferation capabilities, limiting their practical application(Reubinoff, B E et al Nat Biotech 2000; 18:399-404, and Schuldiner, M etal PNAS USA 2000; 97:11307-312). The vasculogenic progenitor cellsisolated by the methods of the present invention are capable ofgenerating large numbers of identical cells by proliferation throughnumerous cell doublings.

The population of vasculogenic progenitor cells isolated according tothe teachings of the present invention is characterized by an abundanceof cells expressing the endothelial progenitor marker VE-cadhedrin(FIGS. 1C-E) and endothelial markers (FIG. 1F), and activelyproliferating, as indicated by incorporation of (BrdU) into the nucleus(FIG. 1I). In the absence of additional stimulus for furtherdifferentiation, these cells are capable of generating large numbers ofmultipotent vasculogenic progenitor cells. In addition, the vasculogenicprogenitor cells may be maintained in a viable state over exceedinglylong periods of time by cryopreservation according to any of the methodsfor conditioning, storage and thawing typically employed in the art(see, for example, U.S. Pat. No. 6,140,123 to Demetriou, et al).

Due to the importance of differentiated cells in various therapeuticapproaches, directed differentiation of embryonic precursor cellspresents an important goal in the art of stem cell culturing. Althoughembryonic stem cells maintained in culture often undergo spontaneousdifferentiation (Thomson J. A. et al Science 1998; 282:1145-47),directed differentiation of embryoid body-derived cells (Shamblott M Jet al PNAS USA 2001; 98:113-18) and human ES cells in coculture with MEFcells (Kaufman D S et al PNAS USA 2001; 98:10716-721) has beendemonstrated by manipulation of environmental factors. For example,Kaufman et al induced hematopoietic differentiation in human ES cells byculture with mouse bone marrow stromal cells (Kaufman D S et al PNAS USA2001; 98:10716-721, and U.S. Pat. No. 6,280,718 to Kaufman, D et al) andCarpenter (US Pat Application No. 20020039724 A1) induced neuronal andglial cell development in neural progenitor cells by exposure to a cAMPactivator and/or neurotrophic growth factor. Benvenisty producedpulsating cardiac muscle cells and neuron-like cells by exposing humanembryoid body cells to a variety of growth factors (Itskovitz-Eldor, Jet al Mol Med 2000; 6:88-95, Schuldiner, M et al PNAS USA 2000;97:11307-312 and International Pat Application No. WO 0210347 A2 toBenvenisty N). However, all of the abovementioned methods employ eithercoculturing, embryoid body formation or selection of progenitors byimmunodetection of cell-surface markers.

Directed differentiation of the vasculogenic progenitor cells of thepresent invention can be effected by exposure to specific vascular,smooth muscle or hematopoietic growth factors.

As is illustrated in the Examples section which follows, exposure of thevasculogenic progenitor cells seeded at a low concentration, to growthfactors, induces differentiation into specific mature cell phenotypes.Exposure to the growth factor hVEGF induced the appearance of bothmorphological and functional indicators of endothelial cell phenotype(FIGS. 1C-F and 2F-H), while exposure to the smooth muscle growth factorhPDGF-BB upregulated smooth muscle cell markers (FIGS. 2A-E). Similarly,exposure to cytokines stimulated hematopoietic differentiation of thevasculogenic progenitor cells (FIGS. 2K-M).

Thus, according to another aspect of the present invention there isprovided a method of preparing somatic cells from the population ofvasculogenic progenitor cells of the present invention, the method iseffected by obtaining a population of vasculogenic progenitor cells asdescribed hereinabove, and culturing the population of vasculogenicprogenitor cells in the presence of at least one growth factor suitablefor inducing somatic cell differentiation.

As used herein, the term “somatic cell” refers to a cell of definitelineage, identifiable as belonging to a specific cell phenotype viamorphological, immunological, biochemical and/or functional criteria.Somatic cells are by definition more differentiated, and lessmultipotent, than progenitor and stem cells. Examples of somatic cells,in the context of the present invention, are endothelial cells, smoothmuscle cells, and blood cells.

Numerous growth factors have been implicated in the complex processes ofvasculogenesis, angiogenesis and hematopoietic differentiation (forreviews, see Carmeliet, P Nature Med 2000; 6:389-95, and Yancopoulos GNature 2000; 407:242-48). Although some (i.e. VEGF, Ang and PDGF) aremore dominant in their effects than others, effective differentiation ofprogenitor cells into somatic cells is typically a result of thecombined, and temporally coordinated action of a number of factors.

Thus, according to one embodiment of this aspect of the presentinvention, directed differentiation is effected by using one or moregrowth factors including, but not limited to, vascular endothelialgrowth factor (VEGF), angiopoietin (Ang), platelet derived growth factor(PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growthfactor (TGF), placental growth factor (PIGF), cytokines, erythropoietin,thrombopoietin, transferrin, insulin, stem cell factor (SCF),Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophagecolony stimulating factor (GM-CSF). Such factors are commerciallyavailable to one skilled in the art, in preparations suitable for use incell culture.

Furthermore, it will be appreciated that the abovementioned growthfactors may comprise families of factors including related moleculeshaving different, and divergent roles in the developmental process.Thus, exposure to members of the VEGF family (for example VEGF-A, VEGF-B. . . VEGF-D), GM-CSF and bFGF may stimulate endothelialdifferentiation, while the PDGF and Ang families are important in smoothmuscle development and lumen formation, respectively.

The differentiation of vasculogenic progenitor cells into vascularsmooth muscle cells (v-SMC) may be directed by increasing aconcentration of serum in the differentiation medium (see in Example 9hereinbelow).

Thus, according to another aspect of the preset invention, there isprovided a method of generating vascular smooth muscle cells fromvasculogenic progenitor cells.

The method is effected by culturing vasculogenic progenitor cells in adifferentiating medium which includes a serum concentration higher than5%, more preferably higher than 9%, most preferably higher than 10%(v/v).

In addition, the differentiation of vasculogenic progenitor cells intoendothelial cells (EC) may be obtained by reducing the concentration ofserum in the differentiation medium (see in Example 9 hereinbelow).

Thus, according to another aspect of the preset invention, there isprovided a method of inducing differentiation of vasculogenic progenitorcells into endothelial cells.

The method is effected by culturing vasculogenic progenitor cells in adifferentiating medium which includes a serum concentration lower than5%, more preferably lower than 3%, most preferably lower than 2% (v/v).

The differentiation, maturation and/or functionality of vasulogeniccells (v-SMC and/or EC) can be further enhanced by exposing thevasculogenic cells to a shear force of at least 1 dyne/cm², preferablyat least 5 dyne/cm², most preferably at least 10 dyne/cm² for a timeperiod sufficient to enhance differentiation, maturation and/orfunctionality of the vasculogenic cells (see Example 8 hereinbelow).Preferably the exposure of vasulogenic cells to a shear force iseffected by using a flow chamber such as illustrated in FIG. 10.

While reducing the present invention to practice, it was revealed thatfollowing initial exposure to differentiation medium and size selectionby filtration, the vasculogenic progenitor cells, and not the smoothmuscle progenitors of the present invention demonstrate robust nuclearuptake of BrdU, indicating cell proliferation.

Thus, according to another aspect of the present invention, there isprovided a cell culture comprising a population of vasculogenicprogenitor cells being sustainable in a proliferative, undifferentiatedstate for as long as 14 days or more and being capable ofdifferentiation into smooth muscle, endothelial and/or hematopoieticcells upon exposure to at least one angiogenic, vasculogenic orhematopoietic growth factor, as detailed hereinabove. Thus, the cellculture of the present invention can be expanded and maintained in arelatively undifferentiated state.

The pluripotent, and proliferative character of embryonic and adult stemcells has naturally been exploited for the benefit of in vitro tissuepreparation and engineering. In tissue engineering, tissue progenitorsor precursors are cultured in vitro with appropriate differentiationfactors, to achieve not only differentiation on the level of theindividual cells, but also morphological, biochemical and anatomicalorganization into recognizable and functional tissue and organstructures, which may be used as a source for tissue/organ grafts, forartificial organ support, or organ-bioreactors. Examples of tissues thathave been engineered in vitro are cartilage (Koch R J and Gorti G KFacial Plast Surg 2002; 18:59-68), skin (Lee K H Yonsei Med J 2000;41:774-79), genitourinary tissues (Atala A Curr Opin Urol 1999;9:517-26) and pancreatic islets (Maria-Engler S S et al Braz J Med BiolRes 2001; 34:691-7). However, these tissues have been engineered fromdifferentiated tissue components, not from stem cells. Embryonic stemcells have been used to produce functional pancreatic islet-likestructures (Lemelsky, et al Science 2001; 292:1389-94) and blood tissue(Kaufman D S et al PNAS USA 2001; 98:10716-21) in vitro.

Vessel-like structures have also been formed in vitro. Kaushal et al(Nat Med 2001; 7:1035-40) reported peripheral endothelial progenitorsforming functional neovessels on decellularized porcine vessels.Levenberg et al (PNAS USA 2002; 99:4391-96), working with human embryoidbody derived endothelial cells, demonstrated formation of tube-likestructures in matrigel, and microvessels upon transplantation. However,these vessel-like structures typically lack the normal complexvascular/mural organization characteristic of normal blood vessels.

While reducing the present invention to practice, it was surprisinglyuncovered that the vasculogenic progenitor cells of the presentinvention form small, capillary-like vessels when grown in matrigel withappropriate growth factors (FIGS. 3A-G), and larger vascular structureson alginate scaffolds (FIGS. 4A and 4B). In both cases, normalendothelial and mural organization were observed (FIGS. 3E-G and 4A-B),as well as blood cell formation within the vascular structures.

Thus, according to another aspect of the present invention, there isprovided a method for preparing vascular tissue. The method is effectedby culturing the population of vasculogenic progenitor cells of thepresent invention in the presence of at least one vasculogenic and/orangiogenic growth factor, under conditions suitable for inducingvascular tissue differentiation.

According to one embodiment of this aspect of the present invention,vascular tissue is prepared by culturing the vasculogenic progenitorcells in a semi-solid, vascularization-promoting medium. Such a mediumtypically comprises extracellular matrix components (for example,Matrigel-B D Biosciences, Bedford, Mass. USA) or collagen (e.g. rat tailcollagen I), in which growth factor-treated, differentiatingvasculogenic cells are mixed following aggregation. The growth factorsmay be any of the abovementioned vasculogenic and/or angiogenic factors,such as vascular endothelial growth factor (VEGF), angiopoietin (Ang),platelet derived growth factor (PDGF), ephrin (Eph), fibroblast growthfactor (FGF), tumor growth factor (TGF) and placental growth factor(PIGF), known to induce vasculogenic and/or angiogenic growth ordevelopment. In a preferred embodiment, the growth factors are 50 ng/mlVEGF₁₆₅ and 10 ng/ml hPDGF-BB. Growth of vascular structures istypically evident after 7-15 days incubation. Characteristic endothelialcell components, such as Weibel-Palade bodies and lipoprotein capsules;vessel lumen, and blood cells are detected by histology and electronmicroscopy, as detailed in the Examples section which follows.

Complex macroscopic tissue architecture may also be mimicked in vitro byseeding the progenitors of the present invention on a porous support, orscaffold. Such supports are well known in the art (see U.S. Pat. Nos.5,759,830 and 5,770,417 to Vacanti et al, and 6,379,962 to Holy et al),and have been recently proposed, for example, as tubular blood vesselprostheses for vascularization and epithelialization by host cells, forvascular regeneration (U.S. Pat Application 20020019663 A1 to Termin, PL et al), for wound repair with fibroblasts (U.S. Pat Application20020076816 to Dai, J et al) and for in vitro bone engineering (U.S. PatApplication No. 20020028511 to deBruijn, J D et al). In one embodimentof the present invention, vascular tissue of greater than capillary sizeis prepared by culturing the vasculogenic progenitor cells on a3-dimensional scaffold. In a preferred embodiment, the scaffold is aporous, biodegradable sponge-like material such as poly-L lactic acid,polylactic-glycolic acid or alginate, and differentiation mediumcontains growth factors VEGF₁₆₅ (50 ng/ml) and hPDGF-BB (10 ng/ml).Vascular tissue of the present invention grown on such an alginatescaffold typically demonstrates vascular characteristics such as lumen,endothelial and smooth muscle cells, cell inclusions and von WillebrandFactor at 14 days in culture (FIGS. 4A-B).

Living vascular tissue prepared by the method of the present inventioncan be used for regenerative therapy, and for neovascularization ofnon-vascular tissue. Vascular tissue may be implanted into embryonic,growing or adult organisms suffering from insufficient or faultyvascularization, as in the microvascular pathology of diabetes, or intotissues experiencing, or at risk of ischemic damage, as in ischemicheart disease and cerebral-vascular disease. Similarly, vascular tissueof the present invention can provide blood vessels of large diameter fortissue replacement therapy in cases of surgical bypass, vasculardegeneration such as atherosclerosis and autoimmune disease.

It will be appreciated that differentiating cultures or vascular tissuesprepared from vasculogenic progenitor cells of the present inventionalso provide a model suitable for the investigation of processeseffecting vascular development and function. For example, the cells andtissues of the present invention may be cultured in the presence ofsuspected toxic materials, antibodies, teratogens, drugs and the like,or exposed to non-standard environmental factors such as temperature,gas partial pressure and pH, or co-cultured in the presence of cellsfrom other tissues or other organisms. Changes in parameters of growthand development, such as failure or delay of endothelial markerexpression, loss of proliferative capacity, or dis-organization of invitro vascularization can be assessed to determine the effect of variousfactors.

Thus, according to another aspect of the present invention, there isprovided a method of determining an effect of a factor on vasculardevelopment, growth and/or modification. The method is effected byexposing the population of vasculogenic progenitor cells of the presentinvention to the factor, and determining an effect of the factor on thecells.

The vasculogenic progenitor cells can be exposed to a factor suspectedof inhibiting or downregulating vascular development, growth ormodification. Such assays are well known in drug development andresearch, and may be employed to test undesirable side effects ofsubstances intended for the treatment of other, non-vascular processes,or, alternatively, may be used to discover novel inhibitors ofvasculogenesis. In order to enable assessment of effects inhibitingvascular development and growth, conditions of culturing thevasculogenic progenitor cells should be favorable, or more preferably,optimal, for vasculogenesis and angiogenesis. This includes optimizationof medium components (such as growth or differentiation factors),temperature, substrate composition, gas partial pressures and the like,for the specific stage of vascular development being investigated.

Indeed, while reducing the present invention to practice, the presentinventors found that incubation of vasculogenic progenitor cells of thepresent invention with an angiogenesis-inhibiting anti VE-cad mAbprevented differentiation by hVEGF (FIGS. 5A-B). Similarly, a drugintended for treatment of early complications of pregnancy could bescreened for potential harmful effects on embryonic vasculardevelopment, by exposing vasculogenic progenitor cells, removing thedrug and monitoring modulation of growth or development of the cells bymethods commonly used in the art. Similarly, factors stimulating orupregulating angiogenesis and/or vasculogenesis in the vasculogenicprogenitor cells can be best assessed under sub-optimal conditions ofculturing. Substances affecting vasculogenesis and/or angiogenesisinclude peptides, peptidomimetics, polypeptides, antibodies, chemicalcompounds and biological agents.

Since progenitor cell populations are highly amenable to tissueengineering, transplantation and regenerative therapy, geneticmanipulation of such cells can provide a source of developing cellpopulations bearing unique, previously unattainable characteristics.

As is clearly illustrated in Example 1 of the Examples sectionhereinbelow, the vasculogenic progenitor cell population of the presentinvention exhibits active proliferation thus making such cells amenableto genetic manipulations rendering such cells, for example, capable ofexpressing at least one exogenous polypeptide. Exogenous polypeptidesexpressed in such a cell culture can be cell surface markers,cell-surface antigens, angiogenic factors, vasculogenic factors andhematopoietic factors. Additional polypeptides that can be expressedare, for example, various receptors, ligands, cell adhesion molecules,enzymes, peptide hormones and immune system proteins.

The vasculogenic progenitor cells of the present invention may bemanipulated to express exogenous polypeptides by introduction of anucleotide sequence encoding the exogenous polypeptide, or a precursorform of the exogenous polypeptide. Exogenous foreign nucleic acidsequences can be transferred to the vasculogenic progenitor cells of theculture by electroporation, calcium phosphate, microinjection,lipofection, retro- or other viral or microbial vector or other meanswell known to one of ordinary skill in the art. Preferably, expressionof the exogenous sequence(s) is inducible. Cells expressing theexogenous polypeptide may be screened and isolated by techniques wellknown in the art including, but not limited to immunoblotting,immunofluorescence, ELISA and RT-PCR. Cells expressing exogenouspolypeptides can be harvested, expanded, differentiated and used for,for example, repairing or augmenting a defect. In this manner, cells,tissues or organs can be prepared with exogenous majorhistocompatability antigens which will decrease rejection oftransplanted materials by the host organism. In addition, cellsexpressing and secreting vasculogenic growth factors, or overexpressinggrowth factor receptors can be selected and cultured, creating culturesof vasculogenic progenitor cells with altered temporal dynamics and/orsensitivities to differentiation factors.

The vasculogenic progenitor cells isolated by the methods of the presentinvention can be used therapeutically, in treatment of vascular andvascular related disease. Potential applications include celltransplantation for repair of damaged and ischemic tissues,vascularization of regenerating tissue and embryonic regenerativemedicine. Examples of such therapeutic applications of stem andprogenitor cells are the augmentation of vessel growth observed in areasof ischemic tissue after implantation of adult endothelial progenitors(Kawamoto A et al Circulation 2001; 103:634-37) and theneovascularization by adult endothelial progenitors following cerebralischemia in induced stroke in mice (Zhang Z G et al Circ Res 2002;90:284-88).

Thus, according to yet another aspect of the present invention there isprovided a method of relieving or preventing a vascular disease orcondition in a mammalian subject. The method is effected byadministering the vasculogenic progenitor cells of the present inventionto the subject.

Methods of administering the progenitor cells of the present inventionto subjects, particularly human subjects include injection orimplantation of the cells into target sites in the subjects, the cellsof the invention can be inserted into a delivery device whichfacilitates introduction by, injection or implantation, of the cellsinto the subjects. Such delivery devices include tubes, e.g., catheters,for injecting cells and fluids into the body of a recipient subject. Ina preferred embodiment, the tubes additionally have a needle, e.g., asyringe, through which the cells of the invention can be introduced intothe subject at a desired location. The progenitor cells of the inventioncan be inserted into such a delivery device, e.g., a syringe, indifferent forms. For example, the cells can be suspended in a solutionor embedded in a support matrix when contained in such a deliverydevice. As used herein, the term “solution” includes a carrier ordiluent in which the cells of the invention remain viable. Carriers anddiluents which can be used with this aspect of the present inventioninclude saline, aqueous buffer solutions, solvents and/or dispersionmedia. The use of such carriers and diluents is well known in the art.The solution is preferably sterile and fluid to the extent that easysyringability exists. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating progenitor cells as described herein in acarrier or diluent and, as required, other ingredients enumerated above,followed by filtered sterilization.

Support matrices in which the vasculogenic progenitor cells can beincorporated or embedded include matrices which are recipient-compatibleand which degrade into products which are not harmful to the recipient.Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, polymeric scaffolds, matrigel and collagenmatrices. Synthetic biodegradable matrices (scaffolds) include syntheticpolymers such as polyanhydrides, polyorthoesters, and polylactic acid.Other examples of synthetic polymers and methods of incorporating orembedding cells into these matrices are known in the art. See e.g., U.S.Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701. These matrices providesupport and protection for the fragile progenitor cells in vivo and are,therefore, the preferred form in which the vasculogenic progenitor cellsare introduced into the recipient subjects.

Differentiation of the implanted vasculogenic progenitor cells of theinvention may be directed by factors originating from the surroundingtissue, or may be initiated by pre-implantation incubation withlineage-specific growth factors. Thus, for example, defects requiringregeneration of smooth muscle can be treated with cells having beenexposed to PDGF-BB, to achieve a population enriched in smooth muscleprecursors.

Vascular disease and conditions that can be treated with the methods ofthe present invention include congenital and acquired vascular disordersand ischemia/reperfusion injury. As used herein, the term “congenitalvascular disorders” refers to vascular disorders existing from birth,including both hereditary and developmental disorders. “Acquiredvascular disorders” refers to vascular disorders ensuing after birth,including secondary vascular manifestations of systemic or otherdisease, such as the microvascular pathologies of diabetes.“Ischemia/reperfusion injury” refers to cell or tissue injury resultingfrom interrupted or diminished blood supply, and the tissue damage,especially the inflammatory response, associated with reestablishingcirculation in ischemic tissues.

Conditions which may benefit from such treatment include ischemicconditions (associated, for example, with myocardial, brain orperipheral vascular ischemia), wound healing, tissue grafting (includingtransplant) and conditions involving endothelial cell growth andproliferation, for example after coronary angioplasty, stenting orrelated procedures, re-endothelialization of arterial grafts, andendothelial regeneration in A-V shunts, e.g. in renal dialysis patients.In view of the complications encountered using porcine progenitorxenografts in primates (Buhler L et al Transplantation 2000; 70:1232-31)the methods of the present invention, which can be applied to human stemcells, are especially suited for treatment of such vascular conditions.

Since it was observed herein that the vasculogenic progenitor cells ofthe invention, when exposed to hematopoietic growth factors andcytokines, can be induced to differentiate into blood cell progenitorsand mature blood cells (FIGS. 2K-M and 3D-F, respectively), thevasculogenic progenitor cells of the invention can also be used fortreating or preventing a hematological disease or condition in amammalian subject.

Such treatment can be effected by administering the cells into a subjectunder conditions suitable for stimulating differentiation into bothendothelial and blood cells. Hematological diseases or conditions thatcan be treated or prevented in this manner include congenital andacquired blood disorders, clotting disorders and neoplastic disease.

One example of a clotting disorder suitable for treatment by the methodof the present invention is von Willebrand's disease, a type ofhemophilia caused by deficiency in the endothelial von Willebrandclotting factor. While reducing the present invention to practice, itwas uncovered that differentiated endothelial cells prepared by themethods of the present invention contain von Willebrand factor (FIGS.3A-G and 4A-B). Thus, endothelial cells or endothelial progenitor cellsof the present invention, or compositions thereof can be administered,producing the clotting factor and alleviating the clotting deficiency.

In addition to the abovementioned therapeutic applications, vasculogenicprogenitor cells isolated and prepared by the methods of the presentinvention can be used to provide vascularization of non-vascular, orinherently poorly vascularized tissue. It will be appreciated that oneof the most important challenges facing the field of tissue engineeringis the adequate perfusion of tissue and organs prepared in vitro forimplantation. To date, most tissue engineering methods have relied onmicroporous supports and vascularization from the host to providepermanent engraftment and transfer of oxygen and nutrients, with varyingand often unpredictable results, especially where thick, complex tissues(e.g. liver) are concerned. One alternative approach is the fabricationof “vascular” channels in silicon by micromachining, for population bymixed hepatocytes and endothelial cells in vitro (Kaihara S et al TissueEng 2000; 6:105-07). In another approach more closely mimicking normaldevelopment, endothelial cells have been cocultured with skin (Black A Fet al Cell Biol Toxicol 1999; 15:81-90) or adipose (Frerich B et al IntJ Oral Maxillofac Surg 2001; 30:414-20) cells to provide a vascularnetwork for the growing tissue. However, none have been successful inengineering viable, implantable vascularized tissues.

Thus, according to a further aspect of the present invention, there isprovided a method of vascularizing a mammalian tissue. The method iseffected by obtaining a population of vasculogenic progenitor cells andcontacting the cells with a mammalian tissue under conditions suitablefor differentiation of the vasculogenic progenitor cells intoendothelial and smooth muscle cells. In one preferred embodiment, themammalian tissue is an engineered, non-vascular tissue.

Examples of such engineered tissue are masses of in vitro preparedhepatocytes, epidermal and dermal cells, pancreatic and bone cells forimplantation. Contacting the tissue with the differentiatingvasculogenic progenitor cells can be by coculture in semisolid matrix oron a porous scaffold, as is commonly used in engineered tissuearchitecture, as detailed hereinabove. Contacting the mammalian tissuecan be performed in vitro, prior to implantation into the host organism,or in vivo, into a previously implanted or existing tissue. In anotherpreferred embodiment, the mammalian tissue is an embryonic tissue,prepared for implantation into adult host organism, or for implantationand growth as an embryo.

While reducing the present invention to practice, it was also observedthat the cells larger than 50 μm retained by the size selection step ofthe present methodology comprise a population enriched in smooth musclecells precursors, expressing characteristic epithelial cell markers andmorphology (FIGS. 1H and 1G, respectively).

Thus, the present invention also provides a method of preparingepithelial progenitor cells from undifferentiated ES cells. The methodis effected by culturing the undifferentiated ES cells in a mannersuitable for differentiation into vasculogenic progenitor cells andisolating cells larger than 50 μm. Conditions for culture of theepithelial precursors, and their differentiation into smooth musclecells, were substantially similar to those detailed herein for thevasculogenic progenitor cells, with substitution of smooth muscle orepithelial growth factors, such as PDGF-BB, in place of endothelial orvasculogenic growth factors. However, it was noted that the epithelialand smooth muscle cells lack the proliferative capacity of the smaller,vasculogenic progenitor cells.

Adult vascular tissue is comprised of endothelial and epithelial cells,distinguishable by size, morphology and cell markers, as well aslocation and function. Current methods for the isolation of vascularcell types rely upon cell surface marker detection, immunofluorescenceand flow cytometry (see, for example, Kevil E G and Bullard D C ActaPhysiol Scand 2001; 173:151-57), making the preparation of vascularcells for experimentation and primary culture cumbersome, expensive andinefficient. Thus, it will be appreciated that the methods of thepresent invention may be employed to isolate and prepare cells fromvascular tissue, as well as from undifferentiated stem cells. In apreferred embodiment, the cells of the vascular tissue are dissociatedby mechanical, or enzymatic means, such as trypsin or collagenasedigestion, to obtain a mixed population of dissociated cells, and thesmaller (smaller than 50 μm) endothelial cells isolated by sizeselection as detailed herein for the vasculogenic progenitor cells.Similarly, adult epithelial cells can be isolated from vascular tissueby a similar method, wherein the retained cell population (greater than50 μm), rich in epithelial cells, is collected.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Materials and Methods

Cell Culture

Undifferentiating human embryonic stem (hES) cells (H9.2, H13) weregrown on inactivated mouse embryonic feeder layer (MEF) as previouslydescribed (Amit M, et al. Dev Biol 2000; 227: 271-78), in 80% knock-outDMEM medium (no pyruvate, high glucose formulation; Life TechnologiesInc., Rockville, Md. USA) supplemented with 20% FBS (HyClone, Logan,Utah, USA), or serum replacement and bFGF, 1 mM L-glutamine, 0.1 mMmercaptoethanol, and 1% nonessential amino acid stock (Life TechnologiesInc., Rockville, Md., USA). hES cells were removed from the feeder layerusing EDTA 5 mM supplemented with 1% fetal bovine serum (FBS; HyClone,Logan, Utah, USA) and dispersed to single-cells using a 40 μm meshstrainer (Benton, Dickinson and Co, Discovery Labware, Bedford, Mass.,USA).

For differentiation, undifferentiated hES single cells were plated ontype IV collagen-coated (Becton Dickinson and Co, San Jose, Calif., USA)or 0.1% gelatin-coated (Sigma Chemical Co., St Louis Mo., USA) 6-welldishes at a concentration of 5×10⁴ cells/cm², in differentiation mediumconsisting of alpha MEM medium (Life Technologies Inc., Rockville, Md.,USA) supplemented with 10% FBS (HyClone, Logan, Utah, USA) and 0.1 mMP-mercapoethanol (Life Technologies Inc., Rockville, Md., USA). On day 6of culture cells were filtered through a 40 μm mesh strainer (Becton,Dickinson and Co, Discovery Labware, Bedford, Mass., USA) and wereanalyzed or recultured for further differentiation. For reculture, thestrained cells were seeded at 2.5×10⁴ cells/cm² on type IV collagencoated dishes (Benton Dickinson and Co, San Jose, Calif., USA) indifferentiation medium (see above) with hVEGF₁₆₅ 50 ng/ml or hPDGF-BB 10ng/ml (both from R&D Systems Inc, Minneapolis, Minn., USA) for anadditional 10-12 days.

Collagen Gel and Matrigel 3-D Vascularization Assays

Before three dimensional culture, filtrated cells cultured for 6 days indifferentiation medium were harvested with EDTA (5 mM) and 0.3-0.5×10⁶cells per ml were incubated in differentiation medium containing 50ng/ml VEGF₁₆₅ and hPDGF-BB 10 ng/ml on uncoated petri dishes (Ein-ShemerIndustries, Israel) for maximum of 24 hours to induce aggregation. Forthe collagen gel assay, aggregates were resuspended in 2×differentiation medium and mixed with an isovolume of rattail collagen I(3 mg/ml) (F.Hoffman-La Roche Ltd, Basel, Switzerland). Initially, 250μl of this mixture was plated in 24-well dishes, which was allowed topolymerize for 15 min at 37° C., before adding 500 μl of differentiationmedium supplemented with the same growth factors. For the Matrigelassay, 24-well dishes were coated with 380 μl of Matrigel (BectonDickinson and Co, San Jose, Calif., USA), incubated 30 min at 37° C.,and aggregates were seeded on the matrigel in differentiation mediumcontaining hVEGF (50 ng/ml) and hPDGF-BB (10 ng/ml). In some assays,aggregates were resuspended within the Matrigel (Becton Dickinson andCo, San Jose, Calif., USA), incubated for 30 min at 37° C., and thenadded to the wells with differentiation medium containing hVEGF (50ng/ml) and hPDGF-BB (10 ng/ml). For all assays, cells were incubated for7-12 days and analyzed using contrast-phase microscope (Olympus OpticalCo Ltd, Hamburg GmbH).

Scaffold Vascularization

LF120 50 μl alginate scaffold (Shapiro L and Cohen S. Biomaterials 1997;18: 583) was kindly provided by Prof Smadar Cohen (Ben GurionUniversity, Beer Sheba, Israel). As described above, the scaffolds wereseeded with 24 hour old ESH cell aggregates prepared as describedhereinabove; approximately 0.5-1.0×10⁶ cells were seeded per scaffold.The cell-containing scaffolds were then cultured in differentiationmedium supplemented with 50 ng/ml VEGF₁₆₅ and 10 ng/ml hPDGF-BB.

Hematopoietic Colony Assay

Hematopoietic progenitor capability was demonstrated by seedingfiltrated, VEGF-treated, VE-cad+ESH cells, as single cells, 1-2×10⁵cells per plate, in semisolid media supplemented with cytokines(Methocult GF+ media; StemCell Technologies, Vancouver BC) (for detailsof the assay, see Kaufman D S et al, PNAS 2001; 98:10716-21). After 14days incubation, the plates were scored for colony-forming units (CFU),according to standard criteria [Eaves C and Lambie, K Atlas of HumanHematopoietic Colonies (1995); StemCell Technologies, Vancouver, BC].

Immunostaining, Dil-Ac-LDL and BrdU Incorporation

Cultured cells were fixed in situ by incubation with 4% paraformaldehyde(Sigma-Aldrich Corp., St Louis, Mo., USA) in phosphate buffered saline(PBS) (Life Technologies Inc., Rockville, Md., USA) for 30 min at roomtemperature. After washing with PBS, cells were stained according tosuppliers instructions with relevant primary antibodies: goat anti humanKDR (R&D Systems Inc, Minneapolis, Minn., USA), mouse anti hCD31, mouseanti hSMA, mouse anti hCalponin, mouse anti h Smooth muscle myosin heavychain (all from DAKO Corp, Carpenteria, Calif., USA), goat anti humanVE-Cadherin (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA),(DAKO Corp, Carpenteria, Calif., USA), mouse anti Smoothelin (CHEMICON,Intn'l, Inc. Temecula, Calif., USA). Controls consisted of cellsincubated with secondary antibodies alone. Immunostained cultures wereexamined and photographed using fluorescence microscopy (Olympus OpticalCo, Ltd. Hamburg, GmbH).

For uptake of Dill-labeled ac-LDL, cultured ESH cells were incubatedwith 10 μg/ml Dill-labeled ac-LDL (Biomedical Technologies Inc.,Stoughton, Mass., USA) for 4 h at 37° C. Following incubation, cellswere washed 3 times with PBS, fixed with 4% paraformaldehyde for 30minutes, examined and photographed using a fluorescent microscope(Olympus Optical Co, Ltd. Hamburg, GmbH).

BrdU incorporation in ESH cultures and differentiating cells wasexamined using a BrdU staining kit (Zymed Labs Inc., South SanFrancisco, Calif., USA) in-situ, according to manufacturersinstructions. Briefly, BrdU solution was diluted 1:100 in culture mediumand added to the cells overnight, followed by two PBS washes, fixationwith 75% ethanol and specific BrdU immunostaining.

Immunophenotype

Cells were characterized using immunofluorescence staining as previouslydescribed (Reubinoff B E et al., Nat Biotech 2001; 19: 1134). Briefly,filter-separated ESH cells were recultured, as described above, indifferentiation medium (alpha MEM, 10% FBS and 0.1 β-mercaptoethanol) ontype IV collagen plates for 12-20 hours, fixed and assayed forexpression of specific cell-type markers with anti human VE-Cadherin(Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), and antihuman KDR (R&D Systems Inc, Minneapolis, Minn., USA). At least onehundred and fifty cells were scored within random fields (×100) for theexpression of each of these markers, and experiments were repeated atleast three times.

Histolomorphology and Immunohistochemical Analysis

Matrigel or collagen gel containing cells (as describe for the Collagengel and Matrigel 3-D vascularization assays hereinabove) were plated asdescribed above onto glass cover slips, in 24-well dishes. Uponcompletion of treatments, the cell-containing gel blocks on cover slipswere fixed in 10% neutral-buffered formalin, dehydrated in graduatedalcohol baths (70%-100%), and embedded in paraffin. Where used, thealginate scaffolds were directly dehydrated in graduated alcohol. Forgeneral histomorphology, 1-8 μm sections were stained withhematoxylin/eosin or toluidine blue. Deparaffinized sections wereimmunostained with the relevant primary antibodies, using LSAB+ stainingkit (DAKO Corp, Carpenteria, Calif., USA) or Cell and Tissue stainingkit (R&D Systems Inc, Minneapolis, Minn., USA) according tomanufacturers instructions. Stained sections were viewed andphotographed microscopically at X100-X400 magnification.

FACS Analysis

Cells expressing the endothelial progenitor markers VEGFR2 (KDR) andVE-cad were detected and quantified from the two size-separated human EScell populations after filtration and separate reculturing on type IVcollagen, as described above. For FACS analysis, ESH filtered cells werewashed in PBS containing 5% FBS, incubated with human VE-Cadherin (SantaCruz Biotechnology, Inc., Santa Cruz, Calif., USA), or human KDR (R&DSystems Inc, Minneapolis, Minn., USA), washed, and incubated 30 min withsuitable second antibodies. Cells were analyzed using a FACSCalibur(Benton Dickinson and Co, San Jose, Calif., USA) with CELLQUESTsoftware. IN both assays, cells reacted with second antibodies onlyserved as controls.

Electron Microscopy

Cell seeded in Matrigel or collagen gel were fixed for one hour in 3%glutaraldehyde, in 0.1M sodium cacodylate and then post-fixed in 1% OsO₄in veronal-acetate buffer for 1 hour. Preparation for electronmicroscopy analysis was performed according to standard procedure at thePathology Department of the Rambam Medical Center, Haifa, Israel.Briefly, the cells were stained with lead-citrate, dehydrated andembedded in Epon resin. Sections were cut at a thickness of 600 Å usinga diamond knife, examined and photographed using a JEM-100SX electronmicroscope.

Reverse Transcription (RT)-PCR Analysis

Total RNA was extracted from progenitors and different lineage cellsusing TriReagent (Sigma-Aldrich Corp., St Louis, Mo., USA) according tothe manufacturer's instructions. Total RNA was quantified by UVspectrophotometry, and 1 μg was used for each RT sample. RNA was reversetranscribed with M-MLV Reverse Transcriptase (Promega Corp., Madison,Wis., USA) and oligo (dT) primers (Promega Corp., Madison, Wis., USA)according to manufacturer's instructions. PCR amplification of theselected transcripts was done with BIOTAQ™ DNA Polymerase (BIOLINE, LtdGmbH Luckenwalde, Germany) using 1 μl of RT product per reaction,according to manufacturers instructions. In some cases MgCl₂concentration (normally 1.5 mM) was calibrated (indicated below). Toensure semi-quantitative results in the RT-PCR assays, the number of PCRcycles for each set of primers was verified to be in the linear range ofthe amplification. In addition, all RNA samples were adjusted to yieldequal amplification of the housekeeping gene GAPDH as an internalstandard. PCR conditions and protocol consisted of: 5 min at 94° C. (hotstart); followed by 30-40 cycles (actual number noted below) of: (a) 94°C. for 30 sec; (b) annealing temperature (Ta, noted below) for 30 sec;and (c) 72° C. for 30 sec, concluding with a final 7-min extension at72° C. at the end. Oligonucleotide-specific conditions were as follows:a-sma, 32 cycles, Ta 60° C. (Yamamura, H et al. Int. J. Cancer (Pred.Oncol.) 1998; 79: 245); calponin, 35 cycles, Ta 60° C. (Yamamura, H etal. Int. J. Cancer (Pred Oncol) 1998; 79: 245); SM-MHC, 35 cycles, Ta62° C., 1 mM MgCl₂ (Boreham et al. Am J Obsetet Gynycol 2001;185:944-52); SM22α, 35 cycles, Ta 60° C. (Yamamura, H et al. Int. J.Cancer (Pred. Oncol.) 1998; 79: 245); caldesmon, 35 cycles, Ta 60° C.(Duplaa, C. et al., Circ Res. 1997; 80:159); GATA2, 35 cycles, Ta 55° C.(Kaufman D S et al PNAS 2001; 98:10716); AC133, 32 cycles, Ta 60° C.(Shamblott M J et al, PNAS 2001; 98:113); Tie2, 35 cycles, Ta 60° C.(Ahmad, S et al, Cancer 2001; 92:1138); CD31, 32 cycles Ta 60° C.(Quarmby, S et al Arterio Thrombo Vas Biol 1999; 19:588-97); Tal1, 40cycles Ta 53° C. (Kaufman D S et al PNAS 2001; 98:10716); GAPDH, 32cycles, Ta 60° C. (Itskovitz-Eldor J et al Mol Med 2000; 6:88).

Oligonucleotide Primers:

For the PCR reactions the following specific oligonucleotide primerswere used:

(a) α-sma: 5′CCAGCTATGTGAAGAAGAAGAGG 3′ (SEQ. ID. NO: 1) (sense) and 5′GTGATCTCCTTCTGCATTCGGT 3′ (SEQ. ID. NO: 2) (antisense). The predictedsize of band is 965 base pairs;

(b) Calponin: 5′ GAGTGTGCAGACGGAACTTCAGCC 3′ (SEQ. ID. NO: 3) (sense)and 5′ GTCTGTGCCCAACTTGGGGTC 3′ (SEQ. ID. NO: 4) (antisense). Thepredicted size of band is 671 base pairs;

(c) SM-MHC: 5′ CTACAGGAGCATGCTGCAGGATCG 3′ (SEQ. ID. NO: 5) and 5′GCTTGCAGAAGCTGCTTCTCCAGC 3′ (SEQ. ID. NO: 6), corresponding tonucleotides 579 (sense) and 758 (antisense), respectively. The predictedsize of band is 179 base pairs;

(d) SM22α: 5′CGCGAAGTGCAGTCCAAAATCG 3′ (SEQ. ID. NO: 7) (sense) and 5′GGGCTGGTTCTTCTTCAATGGGG 3′ (SEQ. ID. NO: 8) (antisense). The predictedsize of band is 928 base pairs;

(e) Caldesmon: 5′ AACAACCTGAAAGCCAGGAGG 3′ (SEQ. ID. NO: 9) and 5′GCTGCTTGTTACGTTTCTGC 3′ (SEQ. ID. NO: 10), corresponding to nucleotides244 (sense) and 792 (antisense), respectively. The predicted size ofband is 530 base pairs;

(f) GATA2: 5′ AGCCGGCACCTGTTGTGCAA 3′ (SEQ. ID. NO: 11) (sense) and 5′TGACTTCTCCTGCATGCACT 3′ (SEQ. ID. NO: 12) (antisense). The predictedsize of band is 242 base pairs;

(g) AC133: 5′ CAGTCTGACCAGCGTGAAAA 3′ (SEQ. ID. NO: 13) (sense) and 5′GGCCATCCAAATCTGTCCTA 3′ (SEQ. ID. NO: 14) (antisense). The predictedsize of band is 200 base pairs;

(h) Tie2: 5′ ATCCCATTTGCAAAGCTTCTGGCTGGC 3′ (SEQ. ID. NO: 15) (sense)and 5′ TGTGAAGCGTCTCACAGGTCCAGGATG 3′ (SEQ. ID. NO: 16) (antisense). Thepredicted size of band is 512 base pairs;

(i) CD31: 5′ CAACGAGAAAATGTCAGA 3′ (SEQ. ID. NO: 17) (sense) and 5′GGAGCCTTCCGTTCTAGAGT 3′ (SEQ. ID. NO: 18) (antisense). The predictedsize of band is 260 base pairs;

(j) Tal1: 5′ ATGGTGCAGCTGAGTCCTCC 3′ (SEQ. ID. NO: 19) (sense) and 5′TCTCATTCTTGCTGAGCTTC 3′ (SEQ. ID. NO: 20) (antisense). The predictedsize of band is 331 base pairs;

(k) GAPDH: 5′ AGCCACATCGCTCAGACACC 3′ (SEQ. ID. NO: 21)

(sense) and 5′ GTACTCAGCGGCCAGCATCG 3′ (SEQ. ID. NO: 22) (antisense).The predicted size of band is 302 base pairs.

Example 1 Isolation and Enrichment of Human Vasculogenic ProgenitorCells from Human Stem Cells

Despite the overwhelming importance of human stem cell technology toresearch and medicine, application of discoveries made in research withnon-human species to human stem cells has been painstakingly difficult,requiring great ingenuity and much effort. While murine embryonic stemcell (mES) lines, for example, retain their pluripotency in culture, andmay be predictably manipulated to differentiate in vitro into cells ofmesodermal, endodermal and ectodermal lineage, in vitro differentiationin human and other primate ES cell lines has been characterized byinconsistency, disorganization, and lack of synchrony, obviatingsuccessful in vitro tissue organization (see, for example, Thompson, etal Curr Top Dev Biol 1998; 38:133-165). In pursuing the isolation ofvasculogenic progenitor cell from human embryonic stem cells, initialhuman ES mesodermal differentiation was attempted in a novel twodimensional (2D) rather than the three dimensional (3D) model commonlyused in the art, based upon the observation that the 3D embryoid bodystructure is not required for mouse stem cell mesodermal differentiation(Nishikawa S-I., et al., Development 1998; 125: 1747).

Undifferentiated human embryonic stem cell line H9.2 and H13 cells weregrown as previously described (Amit M et al Dev Biol 2000; 227:271-78),removed from feeder layer and plated as single cells on type IV collagencoated dishes with differentiation medium as had been described formouse CCE-ES cells (Yamashita J, et al. Nature 2000; 408:92). Previousexperience with murine stem cells indicated that specific cell seedingconcentration is crucial for induction endothelial differentiation.However, seeding human ES cells in the recommended cell concentration(1×10⁴ cells/cm²) resulted in cell death. Therefore, severalcell-seeding concentrations were investigated. Seeding the cells athigher concentrations on a variety of attachment substrates (1.0-1.5×10⁵cells/cm²) resulted in an inconsistent mixed population ofundifferentiated and differentiating cells (data not showed).Surprisingly, seeding cells at low concentration (5-10×10⁴ cells/cm²) ontype IV collagen substrate promoted differentiation that resulted in twodistinct populations of cell types (FIG. 1B). A significant proportionof the cell population comprised smaller flat cells with large nucleiresembling endothelial progenitor morphology (FIG. 1B, arrows)previously recognized in murine cells (Yamashita J, et al. Nature 2000;408:92), while the remainder were large flat cells with obvious fibrousstructure (FIG. 1B, arrowheads).

In order to separate the two cell populations, and isolate the smaller,human vasculogenic cell progenitors, the cells were filtered through a40 μm strainer, segregating the endothelial-like cells from the largeflat cells. To evaluate the proportion of endothelial progenitors in thecultures, the filtered cell populations were characterized by detectionof specific cell-type markers, as previously described for monitoringthe differentiation of neuron progenitors derived from hES cells(Reubinoff B B et al., Nat Biotech 2001; 19:1134). Filtrated cells wereplated, fixed, and analyzed immunologically for the expression of humanvascular endothelial receptor 2 (VEGFR2, KDR), and vascular endothelialcadherin VE-cad, both known to play an important role in mouseendothelial progenitor development (Nishikawa S-I., et al., Development1998; 125: 1747).

Unexpectedly, when the expression of these markers in the twopopulations was quantified by immunodetection and FACS analysis (FIGS.1C-E), a significant proportion of the smaller, endothelial-like cellswere found to express VE-cad (78%, FIG. 1C) and a smaller portionexpressed VEGFR2 (28%, FIG. 1C). When the smaller, filtrated cells wereplated for 12 hours, fixed and analyzed for immunomorphology withfluorescent anti VE-cad antibody (FIGS. 1D-E), significantly greaterexpression of VE-cad was detected (90.55±5.20%), most likely due to thelow fluorescent intensity observed when VE-cad is expressed at thecell-to-cell junctions (FIG. 1E). Trials of a variety of cell-seedingdensities indicated optimal VE-cad expression at 5×10⁴ cells/cm².

When further characterized by RT-PCR amplification of RNA, theendothelial-like cells were found to actively express the endothelialmarkers CD31 and Tie2; AC133/CD133, GATA2 and Tal1, earlyendothelial/hematopoeitic progenitor cell markers (Peichev, M et al.,Blood 2000; 95:952 and Kaufman D S et al PNAS 2001; 98:10716) (FIG. 1F,Filtrated). RT-PCR of RNA from undifferentiated human stem cells (FIG.1F, hES) demonstrated no CD31, Tie2, Tal1 or GATA2 expression, and onlyminimal expression of AC133. Note that the intensities of the GAPDHbands are identical for both the undifferentiated and differentiatedcell populations (FIG. 1F), indicating the specific nature of the changein cell phenotype with differentiation.

Immunofluorescent staining of the larger, excluded cells (FIG. 1G)revealed the existence of epithelioid phenotype smooth muscle cellfeatures (reviewed by Gittenberger-de Groot A. C, et al PNAS 2000;97:11307) and markers (aSMA) undetected in the smaller, filtered cells.When further characterized by RT-PCR amplification of RNA, the larger,excluded cells were found to actively express epitheliod markersCalponin and Caldesmon; smooth muscle actin (SMA), and SM-MHC (FIG. 1H,Retained). RT-PCR of RNA from the smaller, endothelial-like cellsdemonstrated no expression of any of the epitheliod cell markers (FIG.1H, Filtrated). Note that the intensities of the GAPDH bands areidentical for both the cell populations (FIG. 1H), indicating thespecific nature of the change in cell phenotype with differentiation.

When the two cell populations arising from the low-density seeding, andculturing of human stem cells (hES) were assessed for cell proliferationcapability, the BrdU incorporation assay revealed that the epitheliod,excluded large smooth muscle-like cells are unable to proliferate (FIG.11, arrow) while the smaller, endothelial-like progenitor cells clearlyincorporate the stain, indicating retention of proliferative ability(FIG. 1I). Taken together, these results indicate that human stem cells,seeded as single cells and not as Embryoid Bodies, and cultured in vitroon a cell-free, two-dimensional matrix, can give rise to proliferating,endothelial-like progenitor cells, which can be separated by filtrationfrom smooth muscle-like precursors.

Example 2 In Vitro Induction of Endothelial, Smooth Muscle andHematopoietic Cell Differentiation of Human Vasculogenic ProgenitorCells

In order to study the differentiation potential of the vasculogenicprogenitor cells, cells were recultured on type IV collagen coateddishes, at a lower cell seeding concentration (2.5×10⁴ cells/cm²).Smooth muscle cell differentiation was induced by addingplatelet-derived growth factor BB (hPDGF-BB), which has been found toinduce SMC differentiation in murine (mES), but not human stem cells(Gittenberger-de Groot A. C et al PNAS 2000; 97:11307). After 10-12 daysof culture both spindle-like shaped and epithelioid phenotype cells weredetected in the culture, along with a concomitant induction ofexpression smooth muscle cell markers. RT-PCR analysis detectedupregulation of specific smooth muscle markers such as smooth musclea-actin (SMA), smooth muscle myosin heavy chain (SM-MHC), calponin,SM22, and caldesmon (FIG. 2A, v-SMC), notably undetectable in the RNAfrom non-hPDGF-BB treated cells (FIG. 2A, ESH progenitor cells).Immunofluorescent detection of the human smooth muscle cell markerproteins (aSMA, FIG. 2B; smoothelin, a marker of early smooth muscledevelopment, FIG. 2C; SM-MHC FIG. 2D and Calponin FIG. 2E) confirms thecapacity for further in vitro differentiation of human vasculogenicprogenitor cells by exposure to hPDGF-BB.

To test the potential of differentiation to endothelial cells, the humanvasculogenic progenitor cells were exposed to hVEGF₁₆₅, found to beefficient in murine, but not human endothelial cell induction (YamashitaJ, et al. Nature 2000; 408:92). This manipulation resulted in theinduction of endothelial cell-specific markers: continuous expression ofVE-cad and the appearance of von Willebrand Factor (vWF) stored inWeibel-Palade bodies, as detected by immunofluorescence (FIGS. 2F and2G, respectively), Dill-Ac-LDL uptake in more mature cells (FIG. 2H) andeven stress fibers arrangement in some mature cells (FIG. 2I). Mostsignificantly, growth factor-induced differentiation, with eitherhPDGF-BB or hVEGF₁₆₅, did not induce a lineage-specific commitment,i.e.: both endothelial and smooth muscle cell types were observed withadministration of each of the growth factors. Furthermore, BrdUincorporation into the differentiated cells indicated preservation ofproliferative capability in the vascular endothelium growth-factor(VEGF) treated cells (FIG. 2I), and specifically those cells of thesmaller morphology, while cells treated with hPDGF-BB exhibited impairedproliferation ability (FIG. 2J). Hematopoietic capability of theisolated progenitor cells was also demonstrated. When theVE-cad-expressing population of filtrated, vasculogenic progenitor cellswas cultured in a semisolid medium with cytokines, CFUs indicatinghematopoietic colonies (FIGS. 2K-M) were observed. Thus, differentiationof isolated human vasculogenic progenitor cells may be further induced,and controlled, by specific growth factors in vitro, in a cell-freemedium, without lineage-specific commitment or loss of proliferativecapability.

Example 3 In-vitro Vasculogenesis and Blood Cell Formation by ESH Cells

Crucial events characteristic of vasculogenesis have been induced invitro using murine embryonic stem cell-derived embryoid bodies (see, forexample, Feraud O et al Lab Investig 2001; 81: 1661-89), however effortsto emulate vasculogenic processes in vitro using human pluripotent stemcells have been largely unsuccessful. To study the in-vitrovascularization potential of human vasculogenic progenitor (ESH) cellswe used two different 3-dimensional models: type I collagen gel andMatrigel, which have been used to promote 3D vessel-like formation fromendothelial cells (Mardi J A and Pratt B M, B.M.J Cell Biol. 1988;106:1375; Kubota Yet al J Cell Biol 1988; 107:1589).

Aggregation of the ESH cells, in the presence of hVEGF and hPDGF-BBsupplemented differentiation medium, prior to seeding into type Icollagen (FIG. 3A) or on Matrigel (FIG. 3B) clearly induces sproutingand tube-like structures associated with early vasculogenesis andvascularization of both the collagen and Matrigel substrate.Histological sections demonstrate penetration of the endothelial cellsinto the Matrigel, forming a tube-like network structure characteristicof vascular formation (FIG. 3C). Surprisingly, and of great importance,observation under higher magnification reveals blood cells within thesein-vitro cultivated vessels (FIG. 3D, arrow). Electron microscopyfurther reveals well-formed endothelial-specific Weibel-Palade bodies(WP) in the cell cytoplasm, lipoprotein capsules (Li), endothelial cellsforming a lumen (Lu) in the cords and hematopoietic (BC) developmentwithin the vessels formed by endothelial cells (EC) within the Matrigel(M) (FIGS. 3E-3G). These results demonstrate, for the first time,isolated human vasculogenic progenitor cells having the capacity todifferentiate into functional endothelial cells with lipoproteinmetabolism, factor VIII (vWF) production, blood cells, and allcomponents of vascular structures in vitro, under defined conditions.

Example 4 3 Dimensional Scaffold Vascularization

In vitro vascularization of engineered tissues is a critical aspect ofregenerative medicine, crucial for the maintenance of cultured tissueviability before and after implantation. Large-diameter vascularstructures, suitable for implantation, require a supporting framework,e.g. scaffold, for efficient development and growth. Therefore, thetherapeutic potential of human vasculogenic (ESH) progenitor cells wasinvestigated using an in-vitro tissue engineering model, the3-dimensional alginate scaffold, which has been shown to support invitro tissue formation from fibroblasts and hepatocytes (Shapiro L, andCohen S. Biomaterials 1997; 18: 583; and Glicklis R, et al BiotechnolBioeng 2000; 67:344), but not human vasculogenic progenitors.

When human vasculogenic progenitors were aggregated, as described, andseeded within porous alginate scaffolds, distinct vessel formationaround the scaffold pores was observed after 14 days incubation indifferentiation medium supplemented with both hVEGF and hPDGF-BB (FIG.4A, red-staining structures). Higher magnification examination of thevascular wall structure reveals flat, elongated endothelial cellssurrounded by smooth muscle cells, typical of vascular morphology (FIG.4B). Thus, culturing human vasculogenic progenitor (ESH) cells on 3-Dscaffolds demonstrated, for the first time, the capability for directed,in-vitro vasculogenesis with differentiated human stem cells, faithfulto normal angiogenic development.

Example 5 Human Vasculogenic Progenitor Cell Differentiation as a Modelfor Angiogenesis

Recent studies have demonstrated that some murine embryonic stem cell(mES) systems are capable of reproducing key events and chronology ofthe angiogenic process, providing a potentially useful tool with whichto investigate mechanisms of angiogenesis (Feraud, I et al Lab. Invest.2001; 81: 1669). Of further significance was the observation that mEScells derived from VE-cad deficient strains of mice (VE-cad −/−) failedto develop endothelial sprouts. However, only embryoid bodies (mEB), andnot single cells, were capable of initiation of the vasculogenic eventsin vitro.

To investigate whether directed, in-vitro vascular development fromisolated human vasculogenic progenitor cells accurately reflectsphysiological processes of angio- and vasculogenesis, the effect ofinhibitory antibodies was assessed using BV6, a hVE-cad-specificmonoclonal antibody found to inhibit in vitro tube-formation of humanendothelial cells (Corada M et al., Blood 2001; 97: 1679).

Suprisingly, the anti-VE-cad monoclonal exhibited a strong inhibitoryeffect on in vitro vascularization by ESH cells. 7 days after incubationof ESH cells seeded on Matrigel in differentiation medium supplementedwith growth factors, the vessels and network structures typical of earlyvasculogenesis are clearly discernible in the gel (FIG. 5A). Addition of50 μg/ml of the anti hVE-cad antibody BV6 to the medium clearlydisrupted vasculogenesis, inhibiting essential cell sprouting and theformation of tube and network structures (FIG. 5B). Thus, in-vitro,directed differentiation of isolated human vasculogenic progenitor (ESH)cells exhibits sensitivity to known inhibitors of humanangiogenesis-vasculogenesis, and as such, provides a model for studyingand assessing vascular-related effectors and therapies.

Example 6 Enrichment of Vasculogenic Progenitors

Experiments conducted with hES cells seeded at the cell concentrationstaught by Yamashita et al. (Yamashita J, et al Nature 2000; 408: pages92-96) resulted in cell death (data not shown). As such, several 2Ddifferentiation experiments employing different cell seedingconcentrations on gelatin, laminin or type IV collagen coated disheswere devised and conducted.

Materials and Methods

Non-differentiating hES cells (H9.2 passages 29+36-29+60; H13 passages31-57; 16 passages 35-50) were grown on an inactivated mouse embryonicfeeder layer (MEF). All experiments were preformed using lines H9.2 andH13, while progenitor enrichment and characterization experiments werealso effected using the 16 line. hES cells were split using type IVcollagenase, resulting in small aggregates. The cells were treated with5 mM EDTA in PBS, supplemented with 1% (v/v) fetal bovine serum (FBS;HyClone), and separated into individual cell suspensions using a 40μm-mesh strainer (Falcon) to facilitate differentiation studies and FACSanalysis. Undifferentiated hES cell suspensions were plated on type IVcollagen coated dishes (six well, Becton Dickinson) or 0.1% gelatin(Sigma) coated dishes at a cell density of 5×10⁴ cells/cm², in adifferentiation medium composed of alpha MEM medium (Gibco-BRL)supplemented with 10% FBS (HyClone) and 0.1 mM p-mercapoethanol(Gibco-BRL). Following 6 days of culturing, differentiated cells werefiltered through a 40 μm mesh strainer (Falcon) and were analyzed orrecultured for differentiation on type IV collagen coated dishes (BectonDickinson) in a differentiation medium containing hVEGF₁₆₅ 50 ng/ml orhPDGF-BB 10 ng/ml (both from R&D Systems Inc) for 10-12 additional days.

Clonal Analysis

An enriched endothelial cell population was immuno-labeled for FACSanalysis with anti VE-Cadherin-FITC (Santa Cruz) and single cells wereisolated using an IVF micro pipette (Cook). Each cell was plated in awell of a 96 well plate (type IV collagen) in an appropriatedifferentiation medium. Following an hour of culturing, each well wasvisually examined (by both light and fluorescence microscopy) to verifythe number of cells plated. Mechanical single cell isolation was usedrather than FACS, since single hES cell do not typically survive theFACS sorting procedure (data not shown). Following one week of culturingrescued single cells, single cell colonies could be observed. At onemonth post culturing, each colony was digested with type W collagenaseand transferred into a well of a 24 well plate (type IV collagen).Confluent cultures were digested and used either for continuousculturing or in Immunophenotype analysis.

Results

Isolation and Characterization of Endothelial and SMC Progenitors

The present research approach (FIG. 1A) relied upon findings that a 3Dembryoid body structure is not required for the differentiation oflateral mesoderm cells (Yamashita J, et al Nature 2000; 408: 92-96.).

However, attempts to use the cell concentration suggested by Yamashitaet al. (1×10⁵-1.5×10⁵ cells/cm²) resulted in a mixed population whichincludes both undifferentiated colonies and multiple cell typesdifferentiated therefrom (FIG. 6B).

Using type IV collagen coated dishes and lower cell seedingconcentrations (5×10⁴-7×10⁴ hES cells/cm²) enabled the present inventorsto generate a more uniform population which can better serve as a sourcefor specific progenitor populations. The latter population included twotypes of cells, smaller, flat cells with large nuclei similar toendothelial progenitors (Yamashita J, et al Nature 2000; 408: 92-96),and large flat cells with fiber arrangement (FIG. 6C).

Thus, the cell population resultant from the second seeding experiment(i.e., 5×10⁴-7×10⁴ cells/cm² seeded on type IV collagen coated dishes)were used in further experiments attempting to recover specificprogenitor populations.

Since the two dominant subpopulations displayed substantial differencesin size, the population generated according to the teachings of thepresent invention was filtered through a 40 μm filter, thus separatingthe endothelial-like cells from the large flat cells. Analysis of theendothelial-like cell fraction revealed preferential survival of aVE-cad enriched population (˜35%) which was further enriched by repeatedfiltration (˜75%). The endothelial-like cell fraction included cellsexpressing CD31 (˜60% of the cells), and cells expressing Flk-1 (˜30% ofthe cells) (FIG. 6D). RT-PCR analysis which was performed on both cellfractions indicated that the endothelial-like cell fraction did notexpress any v-SMCs markers. Immunophenotype analysis of theendothelial-like cell fraction showed upregulation of the CD34 (17±3%;n=3), Tal1 (75±8%; n=3) and Gata2 (42±10%; n=3) proteins (FIG. 6E) whichwere previously implicated as early endothelial/hematopoietic progenitorcell markers (Kaufman D S et al Proc Natl Acad Sci USA 2001;98:10716-10721; Peichev M, et al Blood 2000; 95:952-958; Robertson S M,et al. Development 2000; 127: 2447-2459).

To evaluate proliferative potential, the cell fractions described abovewere analyzed for BrdU incorporation and Ki-67 expression.Pulse-labeling with BrdU for 12 hours revealed that some of theendothelial progenitor cells incorporated BrdU while none of the SMClike cells exhibited this capability (FIGS. 6Fi-ii). Both isolatedfractions were also tested for the expression of Ki-67, a typicalantigen found in dividing cells. This experiment uncovered that 66±2%(n=3) of the recultured endothelial-like cells expressed Ki67 (FIG. 6Fiii) while inactivated mouse embryonic fibroblasts, which served as acontrol, did not stain for Ki-67 (data not shown).

Lineage Differentiation

Studying the differentiation potential of the enriched progenitorfraction required reculturing on type IV collagen coated dishes, atlower cell seeding concentration (2.5×10⁴ cells/cm²). The potential ofsuch cells to continuously differentiate into endothelial cells wastested in the presence of hVEGF₁₆₅, a well-known mitogen of endothelialcells. The presence of this mitogen induced the uptake of Dil-acetylatedlow-density lipoprotein (Ac-LDL) (FIG. 7A) and production of vonWillebrand Factor (vWF) stored in Weibel-Palade bodies (FIG. 7B). Toinduce v-SMC differentiation, a known ‘recruitment factor’ forpericytes-platelet derived growth factor BB (PDGF-BB) was utilized, thisfactor was proven effective in differentiating mouse ES cells into SMClineage (Yamashita J, et al Nature 2000; 408: 92-96). Following 10-12days of culturing in PDGF-BB, spindle-like cells appeared in theculture. These cells expressed smooth muscle α actin (SMA) (FIG. 7C).Other specific v-SMCs markers such as, smooth muscle myosin heavy chain(SM-MHC), SM22, and caldesmon, were also expressed by these cells asrevealed by RT-PCR analysis (FIG. 7D). These results indicate thepotential of the cells isolated herein to differentiate into an SMCphenotype.

Clonal Analysis

Single VE-cad⁺ cells generated from hES cells were examined in order todetermine whether these cells contain common progenitors for endothelialand mural cells. Single VE-cad⁺ cells isolated from an enrichedvasculogenic population were recultured on a type IV collagen-coated 96well plate. One hour following plating, each well was visually examined(by both light and fluorescence) to verify the number of cells plated.In order to study their differentiation capability, single cells werecultured in differentiation medium supplemented with hVEGF, or withhPDGF-BB. Single cell colonies could be observed following 8 days ofculturing under either culture conditions (FIG. 8Ai). Plating efficiencywas at 8%, lower than that reported for the mES system (Yamashita J, etal Nature 2000; 408: 92-96), indicating the difficulty of culturingsingle hES cells (Amit et al, Dev Biol. 2000; 227:271-278).

Cell cultures supplemented with VEGF predominantly included cells havingendothelial cell morphology (FIG. 8A ii), while PDGF-BB-supplementedcultures predominantly included spindle-like cells resembling v-SMCs(FIG. 8A iii). These spindle-like cells expressed SMA and calponin(FIGS. 8Bi-ii). In the VEGF-supplemented cultures, most of the cellswere characterized by vWF production and a Dil-Ac-LDL metabolism (FIG.8C iii), thus indicating an endothelial cell phenotype. The exposure ofVE-cad⁺ cells to a specific growth factor did not result in total cellcommitment to one lineage. The PDGF-BB supplemented cultures includedcells expressing v-SMC markers and cells classified as VE-cad⁺ cells.

Example 7 Human Vasculature within Mouse

Materials and Methods

In order to examine whether hES-derived vasculogenic progenitor cellscan be used to form vasculature in vivo, alginate scaffolds werepre-seeded with the cells (as described hereinabove) and transplantedsubcutaneously in SCID mice. Non-seeded scaffolds served as negativecontrols. Two weeks following transplantation, scaffolds and surroundingtissues were removed from mice and histologically analyzed.

Results

As can be seen in FIGS. 9A-B vascular tubes which were formed in thecell seeded transplanted scaffolds were substantially thicker than thevascular tubes formed in the control non-seeded scaffolds. Stainingtransplanted scaffolds sections with anti human SMA revealed theformation of functional vasculature of human origin which containedmouse blood flow (FIG. 9C). Similar results were observed followingsubcutaneously injecting matrigel plugs containing hES-derivedvasculogenic progenitor cells.

Example 8 The Effect of Shear Stress on hES-Derived Vasculogenic Cells

Materials and Methods

Human ES-derived vasculogenic cells, predominantly vasculogenic smoothmuscle cells (v-SMCs), were cultured in a flow chamber (as illustratedin FIG. 10) and were exposed to flow-induced shear stress for 24 hours.A closed-loop flow circuit circulated sterile EC-differentiation mediumthrough the assembled flow chamber, which inflicted a steady, laminarshear stress of 10 dynes/cm² acting upon the cells. Each experiment wasaccompanied by a static control construct. Following 24 hr exposure toshear stress the cells were removed from culture and histologicallyanalyzed.

Results

As can be seen in FIGS. 11A-B, the phalloidin expression reveals atypical perpendicular organization indicative of mature functionalvasculogenic cells resulting from the shear stress. In addition, theexpression of a-SMA (a specific marker indicative of early vascularsmooth muscle cells) was also substantially affected by the shearstress. These results indicate that shear stress effectively inducesexpression and organization kinetics of stress fibers, thereby enhancingdifferentiation, maturation and functionality of ES-derived vasculogeniccells.

Example 9 Directed Differentiation of hES Vasculogenic Progenitor Cells

Materials and Methods

Human ES cells were grown on type IV collagen coated plates for six daysas described hereinabove. The resultant vasculogenic progenitor cellswere transferred to differentiating media containing high serum level(10%, v/v) or low serum level (2%, v/v) and incubated at 37° C. Thecultured cells were allowed to proliferate and passage every 5-6 daysroutinely. Following an incubation period of 15 days the cells wereremoved from culture and analyzed by RT-PCR and real-time RT-PCR usingspecific markers indicative of endothelial cells (EC) and vascularsmooth muscle cells (v-SMC). Primer sequences and reaction conditionsused in PCR are described in Table I below.

TABLE 1 Length Reaction Primer Marker (bp) Condition SEQ ID NO: PrimersGene 200 32 cycles, 23 F: TGAAGCCTAGCCTGTCACCT CD34 annealing at 60° C.,24 R: CGCACAGCTGGAGGTCTTAT in 1.5 mM MgCl₂ 331 40 cycles, 25 F:ATGGTGCAGCTGAGTCCTCC Tal-1 annealing at 55° C., 26 R:TCTCATTCTTGCTGAGCTTC in 1.5 mM MgCl₂ 362 35 cycles, 27 F:GGGGGAGGTTGGACTGTAAT Ang1 annealing at 60° C., 28 R:AGGGCACATTTGCACATACA in 1.5 mM MgCl₂ 535 35 cycles, 29 F:GGATCTGGGGAGAGAGGAAC Ang2 annealing at 60° C., 30 R:CTCTGCACCGAGTCATCGTA in 1.5 mM MgCl₂ 512 35 cycles, 15 F:ATCCCATTTGCAAAGCTTCTGGCTGGC Tie2 annealing at 60° C., 16 R:TGTGAAGCGTCTCACAGGTCCAGGATG in 1.5 mM MgCl₂ 596 35 cycles, 31 F:ACGGGATGACCAAGTACAGC VE-cad annealing at 60° C., 32 R:ACACACTTTGGGCTGGTAGG in 1.5 mM MgCl₂ 790 35 cycles, 33 F:CTGGCATGGTCTTCTGTGAAGCA KDR annealing at 60° C., 34 R:AATACCAGTGGATGTGATGGCGG in 1.5 mM MgCl₂ 200 32 cycles, 13 F:CAGTCTGACCAGCGTGAAAA AC133 annealing at 60° C., 14 R:GGCCATCCAAATCTGTCCTA in 1.5 mM MgCl₂ 700 35 cycles, 35 F:GAAGCCAGCTTCCACATAAC VCAM annealing at 60° C., 36 R:AGTGGTGGCCTCGTGAATGG in 1.5 mM MgCl₂ 965 35 cycles,  1 F:CCAGCTATGTGAAGAAGAAGAGG αSMA annealing at 60° C.,  2 R:GTGATCTCCTTCTGCATTCGGT in 1.5 mM MgCl₂ 671 35 cycles,  3 F:GAGTGTGCAGACGGAACTTCAGCC Calpon annealing at 60° C.,  4 R:GTCTGTGCCCAACTTGGGGTC in in 1 mM MgCl₂ 179 35 cycles,  5 F:AAGCCAAGAGCTTGGAAGC SM-MHC annealing at 62° C.,  6 R:TCCTCCTCAGAACCATCTGC in 1 mM MgCl₂ 302 27 cycles, 21 F:AGCCACATCGCTCAGACACC GAPDH annealing at 60° C., 22 R:GTACTCAGCGCCAGCATCG in 1.5 mM MgCl₂ 595 35 cycles, 19 F:CAAGCGGTCGTGAATGACAC mCD31 annealing at 60° C., 20 R:CACTGCCTTGACTGTCTTAAG in 1.5 mM MgCl₂

Results

As can be seen in FIGS. 12-13, high levels of v-SMC markers (a-SMA,calponin and SM-MHC) were detected in cells cultured in high serummedia, while the expression EC markers was substantially downregulated.On the other hand, cells which were cultured in low serum mediaexhibited high levels of EC markers (Tie2, CD31, KDR (VEGFR2), VCAM andVE-Cad, while the expression of v-SMC markers was substantiallydownregulated (FIGS. 12 and 14).

In addition, vasculogenic progenitor cells which were generated onmatrigel coated plates and re-cultured in low serum differentiatingmedium exhibited predominantly vascular smooth muscle cells morphology(FIG. 15A). On the other hand, when re-cultured in high serumdifferentiating medium they proliferated continuously and exhibited highrate of vasculature sprouting of along with intensive tube-like networkof endothelial cells (FIG. 15B).

Hence, the results clearly show that hES-derived vasculogenic progenitorcells can be induced to differentiate into EC or v-SMC by theirculturing in differentiating media including low (12%) or high (10%)serum volume concentrations, respectively.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A method of preparing somatic cells from a population of vasculogenicprogenitor cells, the method comprising: (a) obtaining a population ofvasculogenic progenitor cells; and (b) culturing the population ofvasculogenic progenitor cells in the presence of at least one growthfactor suitable for inducing somatic cell differentiation, therebypreparing the somatic cells.
 2. A method of preparing endothelial cellsfrom vascular tissue, the method comprising: (a) subjecting the vasculartissue to conditions designed for dissociating cells from the vasculartissue, thereby obtaining a mixed population of dissociated cells; and(b) isolating cells smaller than 50 μm from said mixed population ofcells, said cells smaller than 50 μm being endothelial cells.
 3. Themethod of claim 2, wherein said cells smaller than 50 μm are isolated byfiltration, morphometry and/or densitometry.
 4. The method of claim 3,wherein said isolation by filtration is effected via a filter having apore size smaller than 50 μm.
 5. The method of claim 2, wherein saidvascular tissue is human vascular tissue.
 6. A method of preparingepithelial cells from vascular tissue, the method comprising: (a)subjecting the vascular tissue to conditions designed for dissociatingcells from the vascular tissue, thereby obtaining a mixed population ofdissociated cells; and (b) isolating cells larger than 50 μm from saidmixed population of cells, said cells larger than 50 μm being epithelialcells.
 7. The method of claim 6, wherein said cells larger than 50 μmare isolated by filtration, morphometry and/or densitometry.
 8. Themethod of claim 7, wherein said isolation by filtration is effected viaa filter having a pore size smaller than 50 μm.
 9. The method of claim6, wherein said vascular tissue is human vascular tissue.
 10. A methodof generating epithelial cells from vasculogenic progenitor cells,comprising culturing the vasculogenic progenitor cells in adifferentiating medium including a serum volume concentration lower than5% for a time period sufficient to induce differentiation of thevasculogenic progenitor cells into endothelial cells.
 11. The method ofclaim 10, wherein said vasculogenic progenitor cells are derived from EScells.
 12. The method of claim 11, wherein said ES cells are human EScells.
 13. A method of enhancing differentiation, maturation and/orfunctionality of vasculogenic cells, comprising exposing thevasculogenic cells to a shear force of at least 1 dyne/cm² for a timeperiod sufficient to enhance differentiation, maturation and/orfunctionality of the vasculogenic cells.
 14. The method of claim 13,wherein said vasculogenic cells are vascular smooth muscle cells orendothelial cells derived from ES cells.